Stabilized preparations for use in metered dose inhalers

ABSTRACT

Stabilized dispersions are provided for the delivery of a bioactive agent to the respiratory tract of a patient. The dispersions preferably comprise a plurality of perforated microstructures dispersed in a suspension medium that typically comprises a hydrofluoroalkane propellant. As density variations between the suspended particles and suspension medium are minimized and attractive forces between microstructures are attenuated, the disclosed dispersions are particularly resistant to degradation, such as, by settling or flocculation. In particularly preferred embodiments, the stabilized dispersions may be administered to the lung of a patient using a metered dose inhaler.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application, No. 09/218,212, is a continuation of pendingapplication Ser. No. PCT/US98/20615, filed Sep. 29, 1998, which is acontinuation-in-part of application Ser. No. 09/133,848, filed Aug. 14,1998, now abandoned, which is a continuation-in-part of application Ser.No. 09/106,932, filed Jun. 29, 1998, now abandoned, which claimspriority from Provisional Application No. 60/060,337, filed Sep. 29,1997, now abandoned.

FIELD OF THE INVENTION

The present invention generally relates to formulations and methods forthe administration of bioactive agents to a patient via the respiratorytract. More particularly, the present invention relates to methods,systems and compositions comprising relatively stable dispersions ofperforated microstructures in a suspension medium that are preferablyadministered via aerosolization using pulmonary, nasal, or topicalroutes.

BACKGROUND OF THE INVENTION

Targeted drug delivery means are particularly desirable where toxicityor bioavailability of the pharmaceutical compound is an issue. Specificdrug delivery methods and compositions that effectively deposit thecompound at the site of action potentially serve to minimize toxic sideeffects, lower dosing requirements and decrease therapeutic costs. Inthis regard, the development of such systems for pulmonary drug deliveryhas long been a goal of the pharmaceutical industry.

The three most common systems presently used to deliver drugs locally tothe pulmonary air passages are dry powder inhalers (DPIs), metered doseinhalers (MDIs) and nebulizers. MDIs, the most popular method ofinhalation administration, may be used to deliver medicaments in asolubilized form or as a dispersion. Typically MDIs comprise a Freon orother relatively high vapor pressure propellant that forces aerosolizedmedication into the respiratory tract upon activation of the device.Unlike MDIs, DPIs generally rely entirely on the patients inspiratoryefforts to introduce a medicament in a dry powder form to the lungs.Finally, nebulizers form a medicament aerosol to be inhaled by impartingenergy to a liquid solution. More recently, direct pulmonary delivery ofdrugs during liquid ventilation or pulmonary lavage using afluorochemical medium has also been explored. While each of thesemethods and associated systems may prove effective in selectedsituations, inherent drawbacks, including formulation limitations, canlimit their use.

Since the introduction of the metered-dose inhaler in the mid 1950s,inhalation has become the most widely used route of administration ofbronchodilators and steroids locally to the airways of asthmaticpatients. Compared with oral administration of bronchodilators,inhalation via an MDI offers a rapid onset of action and a low incidenceof systemic side effects.

The MDI is dependent on the propulsive force of the propellant systemused in its manufacture. Traditionally, the propellant system hasconsisted of a mixture of chlorofluorocarbons (CFCs) which are selectedto provide the desired vapor pressure and suspension stability.Currently, CFCs such as Freon 11, Freon 12, and Freon 114 are the mostwidely used propellants in aerosol formulations for inhalationadministration. While such systems may be used to deliver solubilizeddrug, the selected bioactive agent is typically incorporated in the formof a fine particulate to provide a dispersion. To minimize or preventthe problem of aggregation in such systems, surfactants are often usedto coat the surfaces of the bioactive agent and assist in wetting theparticles with the aerosol propellant. The use of surfactants in thisway to maintain substantially uniform dispersions is said to “stabilize”the suspensions.

Unfortunately, traditional chlorofluorocarbon propellants are nowbelieved to deplete stratospheric ozone and, as a consequence, are beingphased out. This, in turn, has led to the development of aerosolformulations for pulmonary drug delivery employing so-calledenvironmentally friendly propellants. Classes of propellants which arebelieved to have minimal ozone-depletion potential in comparison withCFCs are perfluorinated compounds (PFCs) and hydrofluoroalkanes (HFAs).While selected compounds in these classes may function effectively asbiocompatible propellants, many of the surfactants that were effectivein stabilizing drug suspensions in CFCs are no longer effective in thesenew propellant systems. As the solubility of the surfactant in the HFAdecreases, diffusion of the surfactant to the interface between the drugparticle and HFA becomes exceedingly slow, leading to poor wetting ofthe medicament particles and a loss of suspension stability. Thisdecreased solubility for surfactants in HFA propellants is likely toresult in decreased efficacy with regard to any incorporated bioactiveagent.

More particularly, medicament suspensions in propellants tend toaggregate rapidly. If the particle size of the suspended material cannotbe regulated and aggregation takes place, the valve orifice of theaerosol container may clog, rendering the dispensing device inoperativeor, if a metering valve is employed, it may be rendered inaccurate. Thisunwanted aggregation or flocculation may lead to improper dosages whichcan lead to undesirable results, particularly in the case of highlypotent, low dose medicaments. Moreover, particle aggregation also leadsto fast creaming or sedimentation of the suspension. The resulting phaseseparation is generally addressed by vigorously shaking the MDI deviceimmediately before use. However, patient compliance is difficult tocontrol and many commercially available suspensions are so unstable thateven slight delays between shaking and use can affect dosage uniformity.

Prior art efforts to overcome the difficulties associated with formingstabilized dispersions using environmentally compatible propellantsgenerally involve the addition of HFA-miscible cosolvents (i.e. ethanol)and/or the inclusion of various surfactant systems. For example, severalattempts have dealt with improving suspension stability by increasingthe solubility of surface-active agents in the HFA propellants. To thisend U.S. Pat. No. 5,118,494, WO 91/11173 and WO 92/00107 disclose theuse of HFA soluble fluorinated surfactants to improve suspensionstability. Mixtures of HFA propellants with other perfluorinatedcosolvents have also been disclosed as in WO 91/04011.

Other attempts at stabilization involved the inclusion of nonfluorinatedsurfactants. In this respect, U.S. Pat. No. 5,492,688 discloses thatsome hydrophilic surfactants (with a hydrophilic/lipophilic balancegreater than or equal to 9.6) have sufficient solubility in HFAs tostabilize medicament suspensions. Increases in the solubility ofconventional nonfluorinated MDI surfactants (e.g. oleic acid, lecithin)can also reportedly be achieved with the use of co-solvents such asalcohols, as set forth in U.S. Pat. Nos. 5,683,677 and 5,605,674, aswell as in WO 95/17195. Unfortunately, as with the prior art cosolventsystems previously discussed, merely increasing the repulsion betweenparticles has not proved to be a very effective stabilizing mechanism innonaqueous dispersions, such as MDI preparations.

In addition to the aforementioned surfactant systems, several otherattempts have been made to provide stabilized dispersions inenvironmentally compatible systems. For example, Canadian PatentApplication No. 2,036,844 describes the use of suspensions comprisingprocaterol encapsulated in thermally denatured albumin. Reportedly, thesuspensions provide for controlled release of the encapsulated agent.Another attempt at providing stable systems is described in CanadianPatent Application No. 2,136,704 which discloses medicinal aerosolformulations comprising spray dried products and a hydrogenatedpropellant. The powders apparently contain low levels of a surfaceactive agent to increase particle repulsion and counterbalanceattractive forces. Similarly, PCT international Publication No. 97/44012describes suspension systems comprising powders incorporating low levelsof a surface active agent to create “appropriate repulsive forces” thatcounterbalance electrostatic attractive forces. Yet another system isdescribed in PCT international Publication No. 97/36574 which discussesthe use of powders in metered dose inhalers. In these systems it appearsthat soluble surfactants are added separately to the systems tostabilize the medicament powders. Each of the aforementioned systems isevidently based on the prior art concept that suspension stability islargely achieved by providing repulsive forces that counterbalance thenatural particulate attractive forces. Despite such attempts, it isclear that no one has been able to develop a broadly applicableformulation approach that is able to meet the demanding criteria of gooddry formulation stability while simultaneously being able to satisfy theever increasing regulatory standards for MDIs.

Accordingly, it is an object of the present invention to provide methodsand preparations that advantageously allow for the efficient delivery ofbioactive agents to the pulmonary air passages of a patient in needthereof

It is a further object of the present invention to provide stabilizedpreparations suitable for aerosolization and subsequent administrationto the pulmonary air passages of a patient in need thereof.

It is still a further object of the present invention to providestabilized dispersions that are compatible for use in a metered doseinhaler and provide reproducible dosing levels over the life of thedevice.

SUMMARY OF THE INVENTION

These and other objects are provided for by the invention disclosed andclaimed herein. To that end, the methods and associated compositions ofthe present invention broadly provide for the improved delivery ofbioactive agents using stabilized preparations. Preferably, thebioactive agents are in a form for administration to a patient via therespiratory tract. More particularly, the present invention provides forthe formation and use of stabilized dispersions (also referred to asstabilized respiratory dispersions) and inhalation systems, includingmetered dose inhalers comprising such dispersions and individualcomponents thereof. Unlike prior art formulations for targeted drugdelivery, the present invention employs novel techniques to reduceattractive forces between the dispersed components and to reduce densitydifferences, thereby retarding degradation of the disclosed dispersionsby flocculation, sedimentation or creaming. As such, the disclosedstable preparations facilitate uniform dose delivery by metered doseinhalers, and allow for more concentrated dispersions.

The stabilized preparations of the present invention provide these andother advantages through the use of hollow and/or porous perforatedmicrostructures that substantially reduce attractive molecular forces,such as van der Waals forces, which dominate prior art dispersionpreparations. In particular, the use of perforated (or porous)microstructures or microparticulates that are permeated or filled by thesurrounding fluid medium, or suspension medium, significantly reducesdisruptive attractive forces between the particles. Moreover, thecomponents of the dispersions may be selected to minimize differences inpolarizabilities (i.e. reduced Hamaker constant differentials) andfurther stabilize the preparation. Unlike formulations comprisingrelatively dense, solid particles or nonporous particles (typicallymicronized), the dispersions of the present invention are substantiallyhomogeneous with only minor differences in density between particlesdefined by the perforated microparticulates and the suspension medium.

In addition to the heretofore unappreciated advantages associated withthe formation of stabilized preparations, the perforated configurationand corresponding large surface area enables the microstructures to bemore easily carried by the flow of gases during inhalation thannon-perforated particles of comparable size. This, in turn, enables theperforated microparticles of the present invention to be carried moreefficiently into the lungs of a patient than non-perforated structuressuch as, micronized particles or relatively nonporous microspheres.

In view of these advantages, the dispersions of the present inventionare particularly compatible with inhalation therapies comprisingadministration of the bioactive preparation to at least a portion of thepulmonary air passages. For the purposes of the present application,these stabilized dispersions intended for pulmonary delivery may betermed respiratory dispersions. In particularly preferred embodiments,such respiratory dispersions comprise an environmentally compatiblepropellant and are used in conjunction with metered dose inhalers toeffectively deliver a bioactive agent to the pulmonary air passages ornasal passages of a patient in need thereof.

Accordingly, in preferred embodiments, the invention provides stablerespiratory dispersions for the pulmonary or nasal delivery of one ormore bioactive agents comprising a suspension medium having dispersedtherein a plurality of perforated microstructures comprising at leastone bioactive agent, wherein said suspension medium comprises at leastone propellant and substantially permeates said perforatedmicrostructures.

For all embodiments of the invention, the perforated microstructures maybe formed of any biocompatible material that provides the physicalcharacteristics necessary for the formation of the stabilizeddispersions. In this regard, the microstructures comprise pores, voids,defects or other interstitial spaces that allow the fluid suspensionmedium to freely permeate or perfuse the particulate boundary, thusreducing, or minimizing density differences between the dispersioncomponents. Yet, given these constraints, it will be appreciated that,any material or configuration may be used to form the microstructurematrix. With regard to the selected materials, it is desirable that themicrostructure incorporates at least one surfactant. Preferably, thissurfactant will comprise a phospholipid or other surfactant approved forpulmonary use. As to the configuration, particularly preferredembodiments of the invention incorporate spray dried hollow microsphereshaving a relatively thin porous wall defining a large internal voidalthough other void containing or perforated structures are contemplatedas well.

Along with the perforated microstructures discussed above, thestabilized dispersions of the present invention further comprise acontinuous phase suspension medium. It is an advantage of the presentinvention that any biocompatible suspension medium having adequate vaporpressure to act as a propellant may be used. Particularly preferredsuspension media are compatible with use in a metered dose inhaler. Ingeneral, suitable propellants for use in the suspension mediums of thepresent invention are those propellant gases that can be liquefied underpressure at room temperature and, upon inhalation or topical use, aresafe, toxicologically innocuous and free of side effects. Further, it isdesirable that the selected suspension medium should be relativelynon-reactive with respect to the suspended perforated microstructures.In this regard, compatible propellants may generally comprisehydrofluoroalkane propellants. Particularly preferred propellantscomprise 1,1,1,2-tetrafluoroethane (CF₃CH₂F) (HFA-134a) and1,1,1,2,3,3,3-heptafluoro-n-propane (CF₃CHFCF₃) (HFA-227),perfluoroethane, monochloroifluoromethane, 1,1 difluoroethane, andcombinations thereof

It will be appreciated that, the present invention further providesmethods for forming stabilized dispersions comprising the steps of:

combining a plurality of perforated microstructures comprising at leastone bioactive agent with a predetermined volume of suspension mediumcomprising at least one propellant to provide a respiratory blendwherein said suspension medium permeates said perforatedmicrostructures; and

mixing said respiratory blend to provide a substantially homogeneousrespiratory dispersion.

As briefly mentioned above (and discussed in more detail below) thestability of the formed dispersions may be flier increased by reducing,or minimizing the Hamaker constant differential between the perforatedmicrostructures and the suspension medium. Those skilled in the art willappreciate that, Hamaker constants tend to scale with refractiveindices. In this regard, the present invention provides preferredembodiments directed to further stabilizing dispersions by reducingattractive van der Waals forces comprising the steps of:

providing a plurality of perforated microstructures; and

combining the perforated microstructures with a suspension mediumcomprising at least one propellant wherein the suspension medium and theperforated microstructures are selected to provide a refractive indexdifferential value of less than about 0.5.

Along with the formation and stabilization of dispersions, the presentinvention is further directed to the pulmonary delivery of at least onebioactive agent using a metered dose inhaler. As used herein, the terms“bioactive agent” refers to a substance which is used in connection withan application that is therapeutic or diagnostic in nature such as,methods for diagnosing the presence or absence of a disease in a patientand/or methods for treating disease in a patient. The bioactive agentmay be incorporated, blended in, coated on or otherwise associated withthe perforated microstructure.

Accordingly, the present invention provides for the use of a propellantin the manufacture of a stabilized dispersion for the pulmonary deliveryof a bioactive agent whereby the stabilized dispersion is aerosolizedusing a metered dose inhaler to provide an aerosolized medicament thatis administered to at least a portion of the pulmonary air passages of apatient in need thereof, said stabilized dispersion comprising asuspension medium having dispersed therein a plurality of perforatedmicrostructures comprising at least one bioactive agent wherein thesuspension medium comprises at least one propellant and substantiallypermeates said perforated microstructures.

Yet another aspect of the invention provides methods for the pulmonarydelivery of one or more bioactive agents comprising the steps of:

providing a pressurized reservoir containing a stabilized respiratorydispersion comprising a suspension medium having dispersed therein aplurality of perforated microstructures comprising one or more bioactiveagents, wherein said suspension medium comprises a propellant andsubstantially permeates said perforated microstructures;

aerosolizing said respiratory dispersion by releasing pressure on thepressurized reservoir to provide an aerosolized medicament comprisingsaid perforated microstructures; and

administering a therapeutically effective amount of said aerosolizedmedicament to at least a portion of the pulmonary passages of a patientin need thereof.

It will be appreciated that, due to the aerodynamic characteristicspreferably afforded by the disclosed perforated microstructures, thepresent invention is particularly efficient at delivering the selectedbioactive agent into the bronchial airways. As such, in another aspect,the invention provides methods for increasing the effective pulmonarydeposition of a bioactive agent using a metered dose inhaler comprisingthe steps of:

associating said bioactive agent with a plurality of perforatedmicrostructures having a mean aerodynamic diameter of less than about 5μm;

dispersing said perforated microstructures in a suspension mediumcomprising a propellant to provide a respiratory dispersion; and

charging a metered dose inhaler with said respiratory dispersion whereinsaid charged metered dose inhaler provides a fine particle fraction ofgreater than approximately 20% w/w upon activation.

With regard to administration, another aspect of the invention isdirected to systems for the administration of one or more bioactiveagents to a patient. In preferred embodiments, the systems comprise ametered dose inhaler. Accordingly, the present invention furtherprovides systems for the pulmonary administation of a bioactive agentcomprising:

a fluid reservoir;

a metering valve operably associated with said fluid reservoir; and

a stabilized dispersion in said fluid reservoir wherein said stabilizeddispersion comprises a suspension medium having dispersed therein aplurality of perforated microstructures comprising at least onebioactive agent wherein said suspension medium comprises at least onepropellant and substantially permeates said perforated microstructures.

As to compatible bioactive agents, those skilled in the art willappreciate that, any therapeutic or diagnostic agent may be incorporatedin the stabilized dispersions of the present invention. For example, thebioactive agent may be selected from the group consisting ofantiallergics, bronchodilators, bronchoconstrictors, pulmonary lungsurfactants, analgesics, antibiotics, leukotriene inhibitors orantagonists, anticholinergics, mast cell inhibitors, antihistamines,antiinflammatories, antineoplastics, anesthetics, anti-tuberculars,imaging agents, cardiovascular agents, enzymes, steroids, geneticmaterial, viral vectors, antisense agents, proteins, peptides andcombinations thereof. As indicated above, the selected bioactive agent,or agents, may be used as the sole structural component of theperforated microstructures. Conversely, the perforated microstructuresmay comprise one or more components (i.e. structural materials,surfactants, excipients, etc.) in addition to the incorporated bioactiveagents. In particularly preferred embodiments, the perforatedmicrostructures will comprise relatively high concentrations ofsurfactant (greater than about 10% w/w) along with the incorporatedbioactive agent(s).

As such, another aspect of the invention provides for respiratorydispersions for the pulmonary delivery of one or more bioactive agentscomprising a suspension medium having dispersed therein a plurality ofmicroparticles comprising greater than about 20% w/w surfactant and atleast one bioactive agent wherein said suspension medium comprises atleast one propellant. Those skilled in the art will appreciate that, dueto their other physiochemical characteristics, the morphology of theincorporated high surfactant particulates may vary without substantiallydestabilizing the dispersion. As such, stabilized dispersions may beformed with such particulates even if they exhibit relatively lowporosity or are substantially solid. That is, while preferredembodiments of the present invention will comprise perforatedmicrostructures or microspheres associated with high levels ofsurfactant, acceptable dispersions may be formed using relatively lowporosity particulates of the same surfactant concentration. In thisrespect, such embodiments are specifically contemplated as being withinthe scope of the present invention.

In addition to the components mentioned above, the stabilizeddispersions may optionally comprise one or more additives to furtherenhance stability or increase biocompatibility. For example, varioussurfactants, co-solvents, osmotic agents, stabilizers, chelators,buffers, viscosity modulators, solubility modifiers and salts can beassociated with the perforated microstructure, suspension medium orboth. The use of such additives will be understood to those of ordinaryskill in the art and the specific quantities, ratios, and types ofagents can be determined empirically without undue experimentation.

Other objects, features and advantages of the present invention will beapparent to those skilled in the art from a consideration of thefollowing detailed description of preferred exemplary embodimentsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A1 to 1F2 illustrate changes in particle morphology as a functionof variation in the ratio of fluorocarbon blowing agent to phospholipid(PFC/PC) present in the spray dry feed. The micrographs, produced usingscanning electron microscopy and transmission electron microscopytechniques, show that in the absence of FCs, or at low PFC/PC ratios,the resulting spray dried microstructures comprising gentamicin sulfateare neither particularly hollow or porous. Conversely, at high PFC/PCratios, the particles contain numerous pores and are substantiallyhollow with thin walls.

FIG. 2 is a scanning electron microscopy image of perforatedmicrostructures comprising cromolyn sodium illustrating a preferredhollow/porous morphology.

FIGS. 3A to 3D are photographs illustrating the enhanced stabilityprovided by the dispersions of the present invention over time ascompared to a commercial cromolyn sodium formulation (Intal,Rhone-Poulenc-Rorer). In the photographs, the commercial formulation onthe left rapidly separates while the dispersion on the right, formed inaccordance with the teachings herein, remains stable over an extendedperiod.

DETAILED DESCRIPTION PREFERRED EMBODIMENTS

While the present invention may be embodied in many different forms,disclosed herein are specific illustrative embodiments thereof thatexemplify the principles of the invention. It should be emphasized thatthe present invention is not limited to the specific embodimentsillustrated.

As set forth above, the present invention provides methods andcompositions that allow for the formation of stabilized suspensions thatmay advantageously be used for the delivery of bioactive agents. Theenhanced stability of the suspensions is primarily achieved by loweringthe van der Waals attractive forces between the suspended particles, andby reducing the differences in density between the suspension medium andthe particles. In accordance with the teachings herein, the increases insuspension stability may be imparted by engineering perforatedmicrostructures which are then dispersed in a compatible suspensionmedium. In this regard, the perforated microstructures preferablycomprise pores, voids, hollows, defects or other interstitial spacesthat allow the fluid suspension medium to freely permeate or perfuse theparticulate boundary. Particularly preferred embodiments compriseperforated microstructures that are both hollow and porous, almosthoneycombed or foam-like in appearance. In especially preferredembodiments the perforated microstructures comprise hollow, porous spraydried microspheres.

With respect to the instant specification, the terms “perforatedmicrostructures” and “perforated microparticles” are used to describeporous products, preferably comprising a bioactive agent, distributedthroughout the suspension medium in accordance with the teachingsherein. Accordingly, the subject terms may be used interchangeablythroughout the instant specification unless the contextual settingindicates otherwise.

When the perforated microstructures are placed in the suspension medium(i.e. propellant), the suspension medium is able to permeate theparticles, thereby creating a “homodispersion”, wherein both thecontinuous and dispersed phases are substantially indistinguishable.Since the defined or “virtual” particles (i.e. those comprising thevolume circumscribed by the microparticulate matrix) are made up almostentirely of the medium in which they are suspended, the forces drivingparticle aggregation (flocculation) are minimized. Additionally, thedifferences in density between the deformed particles and the continuousphase are minimized by having the microstructures filled with themedium, thereby effectively slowing particle creaming or sedimentation.As such, the stabilized suspensions of the present invention areparticularly compatible with inhalation therapies and may be used inconjunction with metered dose inhalers (MDIs), to improve dosereproducibility, reduce clogging of the MDI valve, increase fineparticle fraction, and reduce throat deposition and the resultingside-effects.

Typical prior art suspensions for inhalation therapy comprise solidmicronized particles and small amounts (<1% w/w) of surfactant (e.g.lecithin, Span-85, oleic acid) to increase electrostatic repulsionbetween particles. In sharp contrast, the suspensions of the presentinvention are designed not to increase repulsion between particles, butrather to decrease the attractive forces between particles. Theprincipal forces driving flocculation in nonaqueous media are van derWaals attractive forces. Van der Waals forces are quantum mechanical inorigin, and can be visualized as attractions between fluctuating dipoles(i.e. induced dipole-induced dipole interactions). Dispersion forces areextremely short-range and scale as the sixth power of the distancebetween atoms. When two macroscopic bodies approach one another thedispersion attractions between the atoms sums up. The resulting force isof considerably longer range, and depends on the geometry of theinteracting bodies.

More specifically, for two spherical particles, the magnitude of the vander Waals potential, V_(A), can be approximated by:${V_{A} = {\frac{- A_{eff}}{6H_{0}}\frac{R_{1}R_{2}}{( {R_{1} + R_{2}} )}}},$

where A_(eff) is the effective Hamaker constant which accounts for thenature of the particles and the medium, H₀ is the distance betweenparticles, and R₁ and R₂ are the radii of spherical particles 1 and 2.The effective Hamaker constant is proportional to the difference in thepolarizabilities of the dispersed particles and the suspension medium:A_(eff)=({square root over (A_(Sm)+L )}−{square root over (A_(PART)+L)})², where A_(SM) and A_(PART) are the Hamaker constants for thesuspension medium and the particles, respectively. As the suspendedparticles and the dispersion medium become similar in nature, A_(SM) andA_(PART) become closer in magnitude, and A_(eff) and V_(A) becomesmaller. That is, by reducing the differences between the Hamakerconstant associated with suspension medium and the Hamaker constantassociated with the dispersed particles, the effective Hamaker constant(and corresponding van der Waals attractive forces) may be reduced.

One way to minimize the differences in the Hamaker constants is tocreate a “homodispersion”, that is make both the continuous anddispersed phases essentially indistinguishable as discussed above. Inaddition to exploiting the morphology of the particles to reduce theeffective Hamaker constant, the components of the structural matrix(defining the perforated microstructures) will preferably be chosen soas to exhibit a Hamaker constant relatively close to that of theselected suspension medium. In this respect, one may use the actualvalues of the Hamaker constants of the suspension medium and theparticulate components to determine the compatibility of the dispersioningredients and provide a good indication as to the stability of thepreparation. Alternatively, one could select relatively compatibleperforated microstructure components and suspension mediums usingreadily discernible characteristic physical values that coincide withmeasurable Hamaker constants.

In this respect, it has been found that the refractive index values ofmany compounds tend to scale with the corresponding Hamaker constant.Accordingly, easily measurable refractive index values may be used toprovide a fairly good indication as to which combination of suspensionmedium and particle excipients will provide a dispersion having arelatively low effective Hamaker constant and associated stability. Itwill be appreciated that, since refractive indices of compounds arewidely available or easily derived, the use of such values allows forthe formation of stabilized dispersions in accordance with the presentinvention without undue experimentation. For the purpose of illustrationonly, the refractive indices of several compounds compatible with thedisclosed dispersions are provided in Table I immediately below:

TABLE I Compound Refractive Index HFA-134a 1.172 HFA-227 1.223 CFC-121.287 CFC-114 1.288 PFOB 1.305 Mannitol 1.333 Ethanol 1.361 n-octane1.397 DMPC 1.43  Pluronic F-68 1.43  Sucrose 1.538 Hydroxyethylstarch1.54  Sodium chloride 1.544

Consistent with the compatible dispersion components set forth above,those skilled in the art will appreciate that, the formation ofdispersions wherein the components have a refractive index differentialof less than about 0.5 is preferred. That is, the refractive index ofthe suspension medium will preferably be within about 0.5 of therefractive index associated with the perforated particles ormicrostructures. It will further be appreciated that, the refractiveindex of the suspension medium and the particles may be measureddirectly or approximated using the refractive indices of the majorcomponent in each respective phase. For the perforated microstructures,the major component may be determined on a weight percent basis. For thesuspension medium, the major component will typically be derived on avolume percentage basis. In selected embodiments of the presentinvention the refractive index differential value will preferably beless than about 0.45, about 0.4, about 0.35 or even less than about 0.3.Given that lower refractive index differentials imply greater dispersionstability, particularly preferred embodiments comprise indexdifferentials of less than about 0.28, about 0.25, about 0.2, about 0.15or even less than about 0.1. It is submitted that a skilled artisan willbe able to determine which excipients are particularly compatiblewithout undue experimentation given the instant disclosure. The ultimatechoice of preferred excipients will also be influenced by other factors,including biocompatibility, regulatory status, ease of manufacture,cost.

In contrast to prior art attempts to provide stabilized suspensionswhich require excipients (i.e. surfactants) that are soluble in thesuspension medium, the present invention provides for stabilizeddispersions, at least in part, by immobilizing the bioactive agent(s)and excipients within the structural matrix of the hollow, porousmicrostructures. Accordingly, preferred excipients useful in the presentinvention are substantially insoluble in the suspension medium. Undersuch conditions, even surfactants like, for example, lecithin cannot beconsidered to have surfactant properties in the present invention sincesurfactant performance requires the amphiphile to be reasonably solublein the suspension medium. The use of insoluble excipients also reducesthe potential for particle growth by Ostwald ripening.

As discussed above, the minimization of density differences between theparticles and the continuous phase is largely dependent on theperforated and/or hollow nature of the microstructures, such that thesuspension medium constitutes most of the particle volume. As usedherein, the term “particle volume” corresponds to the volume ofsuspension medium that would be displaced by the incorporatedhollow/porous particles if they were solid, i.e. the volume defined bythe particle boundary. For the purposes of explanation, and as discussedabove, these fluid filled particulate volumes may be referred to as“virtual particles.” Preferably, the average volume of the bioactiveagent/excipient shell or matrix (i.e. the volume of medium actuallydisplaced by the perforated microstructure) comprises less than 70% ofthe average particle volume (or less than 70% of the virtual particle).More preferably, the volume of the microparticulate matrix comprisesless than about 50%, 40%, 30% or even 20% of the average particlevolume. Even more preferably, the average volume of the shell/matrixcomprises less than about 10%, 5% or 3% of the average particle volume.Those skilled in the art will appreciate that, such a matrix or shellvolumes typically contributes little to the virtual particle densitywhich is overwhelmingly dictated by the suspension medium found therein.Of course, in selected embodiments the excipients used to form theperforated microstructure may be chosen so the density of the resultingmatrix or shell approximates the density of the surrounding suspensionmedium.

It will further be appreciated that, the use of such microstructureswill allow the apparent density of the virtual particles to approachthat of the suspension medium substantially eliminating the attractivevan der Waals forces. Moreover, as previously discussed, the componentsof the microparticulate matrix are preferably selected, as much aspossible given other considerations, to approximate the density ofsuspension medium. Accordingly, in preferred embodiments of the presentinvention, the virtual particles and the suspension medium will have adensity differential of less than about 0.6 g/cm³. That is, the meandensity of the virtual particles (as defined by the matrix boundary)will be within approximately 0.6 g/cm³ of the suspension medium. Morepreferably, the mean density of the virtual particles will be within0.5, 0.4, 0.3 or 0.2 g/cm³ of the selected suspension medium. In evenmore preferable embodiments the density differential will be less thanabout 0.1, 0.05, 0.01, or even less than 0.005 g/cm³.

In addition to the aforementioned advantages, the use of hollow, porousparticles allows for the formation of free-flowing dispersionscomprising much higher volume fractions of particles in suspension. Itshould be appreciated that, the formulation of prior art dispersions atvolume fractions approaching close-packing generally results in dramaticincreases in dispersion viscoelastic behavior. Rheological behavior ofthis type is not appropriate for MDI applications. Those skilled in theart will appreciate that, the volume fraction of the particles may bedefined as the ratio of the apparent volume of the particles (i.e. theparticle volume) to the total volume of the system. Each system has amaximum volume fraction or packing fraction. For example, particles in asimple cubic arrangement reach a maximum packing fraction of 0.52 whilethose in a face centered cubic/hexagonal close packed configurationreach a maximum packing fraction of approximately 0.74. For_non-spherical particles or polydisperse systems, the derived values aredifferent. Accordingly, the maximum packing fraction is often consideredto be an empirical parameter for a given system.

Here, it was surprisingly found that the porous structures of thepresent invention do not exhibit undesirable viscoelastic behavior evenat high volume fractions, approaching close packing. To the contrary,they remain as free flowing, low viscosity suspensions having little orno yield stress when compared with analogous suspensions comprisingsolid particulates. The low viscosity of the disclosed suspensions isthought to be due, at least in large part, to the relatively low van derWaals attraction between the fluid-filled hollow, porous particles. Assuch, in selected embodiments the volume fraction of the discloseddispersions is greater than approximately 0.3. Other embodiments mayhave packing values on the order of 0.3 to about 0.5 or on the order of0.5 to about 0.8, with the higher values approaching a close packingcondition. Moreover, as particle sedimentation tends to naturallydecrease when the volume fraction approaches close packing, theformation of relatively concentrated dispersions may further increaseformulation stability.

Although the methods and compositions of the present invention may beused to form relatively concentrated suspensions, the stabilizingfactors work equally well at much lower packing volumes and suchdispersions are contemplated as being within the scope of the instantdisclosure. In this regard, it will be appreciated that, dispersionscomprising low volume fractions are extremely difficult to stabilizeusing prior art techniques. Conversely, dispersions incorporatingperforated microstructures comprising a bioactive agent as describedherein are particularly stable even at low volume fractions.Accordingly, the present invention allows for stabilized dispersions,and particularly respiratory dispersions, to be formed and used atvolume fractions less than 0.3. In some preferred embodiments, thevolume fraction is approximately 0.0001-0.3, more preferably 0.001-0.01.Yet other preferred embodiments comprise stabilized suspensions havingvolume fractions from approximately 0.01 to approximately 0.1.

The perforated microstructures of the present invention may also be usedto stabilize dilute suspensions of micronized bioactive agents. In suchembodiments the perforated microstructures may be added to increase thevolume fraction of particles in the suspension, thereby increasingsuspension stability to creaming or sedimentation. Further, in theseembodiments the incorporated microstructures may also act in preventingclose approach (aggregation) of the micronized drug particles. It shouldbe appreciated that, the perforated microstructures incorporated in suchembodiments do not necessarily comprise a bioactive agent. Rather, theymay be formed exclusively of various excipients, including surfactants.

As indicated throughout the instant specification, the dispersions ofthe present invention are preferably stabilized. In a broad sense, theterm “stabilized dispersion” will be held to mean any dispersion thatresists aggregation, flocculation or creaming to the extent required toprovide for the effective delivery of a bioactive agent. While thoseskilled in the art will appreciate that there are several methods thatmay be used to assess the stability of a given dispersion, a preferredmethod for the purposes of the present invention comprises determinationof creaming or sedimentation time. In this regard, the creaming timeshall be defined as the time for the suspended drug particulates tocream to ½ the volume of the suspension medium. Similarly, thesedimentation time may be defined as the time it takes for theparticulates to sediment in ½ the volume of the liquid medium. Onerelatively simple way to determine the creaming time of a preparation isto provide the particulate suspension in a sealed glass vial. The vialsare agitated or shaken to provide relatively homogeneous dispersionswhich are then set aside and observed using appropriate instrumentationor by visual inspection. The time necessary for the suspendedparticulates to cream to ½ the volume of the suspension medium (i.e., torise to the top half of the suspension medium), or to sediment within ½the volume (i.e., to settle in the bottom ½ of the medium), is thennoted. Suspension formulations having a creaming time greater than 1minute are preferred and indicate suitable stability. More preferably,the stabilized dispersions comprise creaming times of greater than 1, 2,5, 10, 15, 20 or 30 minutes. In particularly preferred embodiments, thestabilized dispersions exhibit creaming times of greater than about 1,1.5, 2, 2.5, or 3 hours.

Substantially equivalent periods for sedimentation times are indicativeof compatible dispersions.

Regardless of the ultimate composition or precise creaming time, thestabilized respiratory dispersions of the present invention preferablycomprise a plurality of perforated microstructures, or microparticulatesthat are dispersed or suspended in the suspension medium.

In such cases, the perforated microstructures comprise a structuralmatrix that exhibits, defines or comprises voids, pores, defects,hollows, spaces, interstitial spaces, apertures, perforations or holesthat allows the surrounding suspension medium to freely permeate, fillor pervade the microstructure. The absolute shape (as opposed to themorphology) of the perforated microstructure is generally not criticaland any overall configuration that provides the desired stabilizationcharacteristics is contemplated as being within the scope of theinvention. Accordingly, preferred embodiments can comprise approximatelymicrospherical shapes. However, collapsed, deformed or fracturedparticulates are also compatible. With this caveat, it will beappreciated that, particularly preferred embodiments of the inventioncomprise spray dried hollow, porous microspheres.

In order to maximize dispersion stability and optimize distribution uponadministration, the mean geometric particle size of the perforatedmicrostructures is preferably about 0.5-50 μm, more preferably 1-30 μm.It will be appreciated that, large particles (i.e. greater than 50 μm)should not be used as large particles may tend to aggregate, separatefrom the suspension and clog the valve or orifice of the container. Inespecially preferred embodiments, the mean geometric particle size (ordiameter) of the perforated microstructures is less than 20 μm or lessthan 10 μm. More preferably, the mean geometric diameter is less thanabout 5 μm, and even more preferably, less than about 2.5 μm. Inespecially preferred embodiments, the perforated microstructures willcomprise a powder of dry, hollow, porous microspherical shells ofapproximately 1 to 10 μm in diameter, with shell thicknesses ofapproximately 0.1 μm to approximately 0.5 μm. It is a particularadvantage of the present invention that the particulate concentration ofthe dispersions and structural matrix components can be adjusted tooptimize the delivery characteristics of the selected particle size.

As discussed throughout the instant specification, the porosity of themicrostructures may play a significant part in establishing dispersionstability. In this respect, the mean porosity of the perforatedmicrostructures may be determined through electron microscopy coupledwith modern imaging techniques. More specifically, electron micrographsof representative samples of the perforated microstructures may beobtained and digitally analyzed to quantify the porosity of thepreparation. Such methodology is well known in the art and may beundertaken without undue experimentation.

For the purposes of the present invention, the mean porosity (i.e. thepercentage of the particle surface area that is open to the interiorand/or a central void) of the perforated microstructures may range fromapproximately 0.5% to approximately 80%. In more preferred embodiments,the mean porosity will range from approximately 2% to approximately 40%.Based on selected production parameters, the mean porosity may begreater than approximately, 2%, 5%, 10%, 15%, 20%, 25% or 30% of themicrostructure surface area. In other embodiments, the mean porosity ofthe microstructures may be greater than about 40%, 50%, 60%, 70% or even80%. As to the pores themselves, they typically range in size from about5 nm to about 400 nm, with mean pore sizes preferably in the range offrom about 20 nm, to about 200 nm. In particularly preferredembodiments, the mean pore size will be in the range of from about 50 nmto about 100 nm. As may be seen in FIGS. 1A1 to 1F2, and discussed inmore detail below, it is a significant advantage of the presentinvention that the pore size and porosity may be closely controlled bycareful selection of the incorporated components and productionparameters.

Along with the geometric configuration, the perforated or porous and/orhollow design of the microstructures also plays an important role in theresulting aerosol properties upon activation of the MDI. In thisrespect, the perforated structure and relatively high surface area ofthe dispersed microparticles enables them to be carried along in theflow of gases during inhalation with greater ease for longer distancesthan non-perforated particles of comparable size. Because of their highporosity, the density of the particles is significantly less than 1.0g/cm³, typically less than 0.5 g/cm³, more often on the order of 0.1g/cm³, and as low as 0.01 g/cm³. Unlike the geometric particle size, theaerodynamic particle size, d_(aer), of the perforated microstructuresdepends substantially on the particle density, ρ: d_(aer)=d_(geo)ρ,where d_(geo), is the geometric diameter. For a particle density of 0.1g/cm³, d_(aer), will be roughly three times smaller than d_(geo),leading to increased particle deposition into the peripheral regions ofthe lung and correspondingly less deposition in the throat. In thisregard, the mean aerodynamic diameter of the perforated microstructuresis preferably less than about 5 μm, more preferably less than about 3μm, and, in particularly preferred embodiments, less than about 2 μm.Such particle distributions will act to increase the deep lungdeposition of the administered agent.

As will be shown subsequently in the Examples, the particle sizedistribution of the aerosol formulations of the present invention aremeasurable by conventional techniques such as, for example, cascadeimpaction or by time of flight analytical methods. Determination of theemitted dose in pressurized inhalations was done according to theproposed U.S. Pharmacopeia method (Pharmacopeial Previews, 22(1996)3065) which is incorporated herein by reference. These and relatedtechniques enable the “fine particle fraction” of the aerosol, whichcorresponds to those particulates that are likely to effectivelydeposited in the lung, to be calculated. As used herein the phrase “fineparticle fraction” refers to the percentage of the total amount ofactive medicament delivered per actuation from the mouthpiece ontoplates 2-7 of an 8 stage Andersen cascade impactor. Based on suchmeasurements, the formulations of the present invention will preferablyhave a fine particle fraction of approximately 20% or more by weight ofthe perforated microstructures (w/w). More preferably, they will exhibita fine particle fraction of from about 25% to 80% w/w, and even morepreferably from about 30 to 70% w/w. In selected embodiments the presentinvention will preferably comprise a fine particle fraction of greaterthan about 30%, 40%, 50%, 60%, 70% or 80% by weight

Further, it has also been found that the formulations of the presentinvention exhibit relatively low deposition rates, when compared withprior art preparations, on the induction port and onto plates 0 and 1 ofthe impactor. Deposition on these components is linked with depositionin the throat in humans. More specifically, commercially available CFCinhalers have simulated throat depositions of approximately 40-70% (w/w)of the total dose, while the formulations of the present inventiontypically deposit less than about 20% w/w. Accordingly, preferredembodiments of the present invention have simulated throat depositionsof less than about 40%, 35%, 30%, 25%, 20%, 15% or even 10% w/w. Thoseskilled in the art will appreciate that, significant decrease in throatdeposition provided by the present invention will result in acorresponding decrease in associated local side-effects such as, throatirritation and candidiasis.

With respect to the advantageous deposition profile provided by theinstant invention, it is well known that MDI propellants typically forcesuspended particles out of the device at a high velocity towards theback of the throat. Since prior art formulations typically contain asignificant percentage of large particles and/or aggregates, as much astwo-thirds or more of the emitted dose may impact the throat. Yet, asdiscussed above, the stabilized dispersions of the present inventionresult in surprisingly low throat deposition upon administration. Whilenot wishing to be bound by any particular theory, it appears that thereduced throat deposition provided by the instant invention results fromdecreases in particle aggregation and from the hollow and/or porousmorphology of the incorporated microstructures. That is, the hollow andporous nature of the dispersed microstructures slows the velocity ofparticles in the propellant stream, just as a hollow/porous whiffle balltravels slower than a baseball. Thus, rather than impacting and stickingto the back of the throat, the relatively slow traveling particles aresubject to inhalation by the patient. Accordingly, a substantiallyhigher percentage of the administered bioactive agent is deposited inthe pulmonary air passages where it may be efficiently absorbed.

Whatever configuration and/or size distribution is ultimately selectedfor the perforated microstructure, the composition of the definingstructural matrix may comprise any one of a number of biocompatiblematerials. It will be appreciated that, as used herein, the terms“structural matrix” or “microstructure matrix” are equivalent and shallbe held to mean any solid material forming the perforatedmicrostructures which define a plurality of voids, apertures, hollows,defects, pores, holes, fissures, etc. that promote the formation ofstabilized dispersions as explained above. The structural matrix may besoluble or insoluble in an aqueous environment. In preferredembodiments, the perforated microstructure defined by the structuralmatrix comprises a spray dried hollow porous microsphere incorporatingat least one surfactant. For other selected embodiments the particulatematerial may be coated one or more times with polymers, surfactants orother compounds which aid suspension.

More generally, the perforated microstructures may be formed of anybiocompatible material that is relatively stable and preferablyinsoluble with respect to the selected suspension medium and can providethe necessary perforated configuration. While a wide variety ofmaterials may be used to form the particles, in particularly preferredembodiments, the structural matrix is associated with, or comprises, asurfactant such as, a phospholipid or fluorinated surfactant. Althoughnot required, the incorporation of a compatible surfactant can improvethe stability of the respiratory dispersions, increase pulmonarydeposition and facilitate the preparation of the suspension. Moreover,by altering the components, the density of the structural matrix may beadjusted to approximate the density of the surrounding medium andfurther stabilize the dispersion. Finally, as will be discussed infurther detail below, the perforated microstructures preferably compriseat least one bioactive agent.

As set forth above, the perforated microstructures of the presentinvention may optionally be associated with, or comprise, one or moresurfactants. Moreover, miscible surfactants may optionally be combinedwith the suspension medium liquid phase. It will be appreciated by thoseskilled in the art that the use of surfactants, while not necessary topractice the instant invention, may further increase dispersionstability, simplify formulation procedures or increase bioavailabilityupon administration. With respect to MDIs, surfactants flirter serve tolubricate the metering valve, thereby ensuring consistentreproducibility of valve actuation and accuracy of dose dispersed. Ofcourse combinations of surfactants, including the use of one or more inthe liquid phase and one or more associated with the perforatedmicrostructures are contemplated as being within the scope of theinvention. By “associated with or comprise” it is meant that thestructural matrix or perforated microstructure may incorporate, adsorb,absorb, be coated with or be formed by the surfactant

In a broad sense, surfactants suitable for use in the present inventioninclude any compound or composition that aids in the formation andmaintenance of the stabilized respiratory dispersions by forming a layerat the interface between the structural matrix and the suspensionmedium. The surfactant may comprise a single compound or any combinationof compounds, such as in the case of co-surfactants. Particularlypreferred surfactants are substantially insoluble in the propellant,nonfluorinated, and selected from the group consisting of saturated andunsaturated lipids, nonionic detergents, nonionic block copolymers,ionic surfactants, and combinations of such agents. It should beemphasized that, in addition to the aforementioned surfactants, suitable(i.e. biocompatible) fluorinated surfactants are compatible with theteachings herein and may be used to provide the desired stabilizedpreparations.

Lipids, including phospholipids, from both natural and synthetic sourcesare particularly compatible with the present invention and may be usedin varying concentrations to form the structural matrix. Generally,compatible lipids comprise those that have a gel to liquid crystal phasetransition greater than about 40° C. Preferably, the incorporated lipidsare relatively long chain (i.e. C,₆-C₂₂) saturated lipids and morepreferably comprise phospholipids. Exemplary phospholipids useful in thedisclosed stabilized preparations comprise egg phosphatidylcholine,dilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidyl-choline, disteroylphosphatidylcholine,short-chain phosphatidylcholines, phosphatidylethanolamine,dioleylphosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, glycolipids, gangliosideGM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearingpolymer chains such as, polyethylene glycol, chitin, hyaluronic acid, orpolyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, andpolysaccharides; fatty acids such as palmitic acid, stearic acid, andoleic acid; cholesterol, cholesterol esters, and cholesterolhemisuccinate. Due to their excellent biocompatibility characteristics,phospholipids and combinations of phospholipids and poloxamers areparticularly suitable for use in the stabilized dispersions disclosedherein.

Compatible nonionic detergents comprise: sorbitan esters includingsorbitan trioleate (Spans® 85), sorbitan sesquioleate, sorbitanmonooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitanmonolaurate, and polyoxyethylene (20) sorbitan monooleate, oleylpolyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, laurylpolyoxyethylene (4) ether, glycerol esters, and sucrose esters. Othersuitable nonionic detergents can be easily identified using McCutcheon'sEmulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.) which isincorporated herein in its entirety. Preferred block copolymers includediblock and triblock copolymers of polyoxyethylene and polyoxypropylene,including poloxamer 188 (Pluronic® F68), poloxamer 407 (Pluronic®F-127), and poloxamer 338. Ionic surfactants such as sodiumsulfosuccinate, and fatty acid soaps may also be utilized. In preferredembodiments, the microstructures may comprise oleic acid or its alkalisalt.

In addition to the aforementioned surfactants, cationic surfactants orlipids are preferred especially in the case of delivery or RNA or DNA.Examples of suitable cationic lipids include: DOTMA,N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride;DOTAP,1,2-dioleyloxy-3-(trimethylammonio)propane;and DOTB, 1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol.Polycationic amino acids such as polylysine, and polyarginine are alsocontemplated.

Those skilled in the art will further appreciate that, a wide range ofsurfactants may optionally be used in conjunction with the presentinvention. Moreover, the optimum surfactant or combination thereof for agiven application can readily be determined by empirical studies that donot require undue experimentation. It will further be appreciated that,the preferred insolubility of any incorporated surfactant in thesuspension medium will dramatically decrease the associated surfaceactivity. As such, it is arguable as to whether these materials havesurfactant-like character prior to contracting an aqueous bioactivesurface (e.g. the aqueous hypophase in the lung). Finally, as discussedin more detail below, surfactants comprising the porous particles mayalso be useful in the formation of precursor oil-in-water emulsions(i.e. spray drying feed stock) used during processing to form thestructural matrix.

Unlike prior art formulations, it has surprisingly been found that theincorporation of relatively high levels of surfactants (i.e.phospholipids) may be used to increase the stability of the discloseddispersions. That is, on a weight to weight basis, the structural matrixof the perforated microstructures may comprise relatively high levels ofsurfactant. In this regard, the perforated microstructures willpreferably comprise greater than about 1%, 5%, 10%, 15%, 18%, or even20% w/w surfactant. More preferably, the perforated microstructures willcomprise greater than about 25%, 30%, 35%, 40%, 45%, or 50% w/wsurfactant. Still other exemplary embodiments will comprise perforatedmicrostructures wherein the surfactant or surfactants are present atgreater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 95%w/w. In selected embodiments the perforated microstructures willcomprise essentially 100% w/w of a surfactant such as a phospholipid.Those skilled in the art will appreciate that, in such cases, thebalance of the structural matrix (where applicable) will preferablycomprise a bioactive agent or non surface active excipients oradditives.

While such surfactant levels are preferably employed in perforatedmicrostructures, they may be used to provide stabilized systemscomprising relatively nonporous, or substantially solid, particulates.That is, while preferred embodiments will comprise perforatedmicrostructures or microspheres associated with high levels ofsurfactant, acceptable dispersions may be formed using relatively lowporosity particulates of the same surfactant concentration (i.e. greaterthan about 10% or 20% w/w). In this respect, such embodiments arespecifically contemplated as being within the scope of the presentinvention.

In other preferred embodiments of the invention, the structural matrixdefining the perforated microstructure optionally comprises synthetic ornatural polymers or combinations thereof In this respect, usefulpolymers comprise polylactides, polylactide-glycolides, cyclodextrins,polyacrylates, methylcellulose, carboxymethylcellulose, polyvinylalcohols, polyanhydrides, polylactams, polyvinyl pyrrolidones,polysaccharides (dextrans, starches, chitin, chitosan, etc.), hyaluronicacid, proteins, (albumin, collagen, gelatin, etc.). Those skilled in theart will appreciate that, by selecting the appropriate polymers, thedelivery profile of the respiratory dispersion may be tailored tooptimize the effectiveness of the bioactive agent.

In addition to the aforementioned polymer materials and surfactants, itmay be desirable to add other excipients to an aerosol formulation toimprove microsphere rigidity, drug delivery and deposition, shelf-lifeand patient acceptance. Such optional excipients include, but are notlimited to: coloring agents, taste masking agents, buffers, hygroscopicagents, antioxidants, and chemical stabilizers. Further, variousexcipients may be incorporated in, or added to, the particulate matrixto provide structure and form to the perforated microstructures (i.e.microspheres). These excipients may include, but are not limited to,carbohydrates including monosaccharides, disaccharides andpolysaccharides. For example, monosaccharides such as, dextrose(anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol,sorbose and the like; disaccharides such as, lactose, maltose, sucrose,trehalose, and the like; trisaccharides such as, raffinose and the like;and other carbohydrates such as, starches (hydroxyethylstarch),cyclodextrins and maltodextrins. Amino acids are also suitableexcipients with glycine preferred. Mixtures of carbohydrates and aminoacids are further held to be within the scope of the present invention.The inclusion of both inorganic (e.g. sodium chloride, calciumchloride), organic salts (e.g. sodium citrate, sodium ascorbate,magnesium gluconate, sodium gluconate, tromethamine hydrochloride) andbuffers is also contemplated.

Yet other preferred embodiments include perforated microstructures thatmay comprise, or may be coated with, charged species that prolongresidence time at the point of contact or enhance penetration throughmucosae. For example, anionic charges are known to favor mucoadhesionwhile cationic charges may be used to associate the formedmicroparticulate with negatively charged bioactive agents, such asgenetic material. The charges may be imparted through the association orincorporation of polyanionic or polycationic materials such aspolyacrylic acids, polylysine, polylactic acid and chitosan.

In addition to, or instead of, the components discussed above, theperforated microstructures will preferably comprise at least onebioactive agent. As used herein, “bioactive agent” refers to a substancewhich is used in connection with an application that is therapeutic ordiagnostic in nature, such as, methods for diagnosing the presence orabsence of a disease in a patient and/or in methods for treating adisease in a patient. Particularly preferred bioactive agents for use inaccordance with the invention include anti-allergics, peptides andproteins, bronchodilators and anti-inflammatory steroids for use in thetreatment of respiratory disorders such as asthma by inhalation therapy.

It will be appreciated that, the perforated microstructures of thepresent invention may exclusively comprise one or more bioactive agents(i.e. 100% w/w). However, in selected embodiments the perforatedmicrostructures may incorporate much less bioactive agent depending onthe activity thereof. Accordingly, for highly active materials theperforated microstructures may incorporate as little as 0.001% by weightalthough a concentration of greater than about 0.1% w/w is preferred.Other embodiments of the invention may comprise greater than about 5%,10%, 15%, 20%, 25%, 30% or even 40% w/w bioactive agent. Still morepreferably, the perforated microstructures may comprise greater thanabout 50%, 60%, 70%, 75%, 80% or even 90% w/w bioactive agent. Inparticularly preferred embodiments, the final stabilized respiratorydispersion desirably contains from about 40%-60% w/w, more preferably50%-70% w/w, and even more preferably 60%-90% w/w of bioactive agentrelative to the weight of the microparticulate matrix. The preciseamount of bioactive agent incorporated in the stabilized dispersions ofthe present invention is dependent upon the agent of choice, therequired dose, and the form of the drug actually used for incorporation.Those skilled in the art will appreciate that, such determinations maybe made by using well-known pharmacological techniques in combinationwith the teachings of the present invention.

Accordingly, bioactive agents that may be administered in the form ofaerosolized medicaments in conjunction with the teachings herein includeany drug that may be presented in a form which is relatively insolublein the selected propellant and subject to pulmonary uptake inphysiologically effective amounts. Compatible bioactive agents comprisehydrophilic and lipophilic respiratory agents, bronchodilators,antibiotics, antivirals, pulmonary lung surfactants,anti-inflammatories, steroids, antihistaminics, leukotriene inhibitorsor antagonists, anticholinergics, antineoplastics, anesthetics, enzymes,cardiovascular agents, genetic material including DNA and RNA, viralvectors, immunoactive agents, imaging agents, vaccines,immunosuppressive agents, peptides, proteins and combinations thereof.Particularly preferred bioactive agents for administration usingaerosolized medicaments in accordance with the present invention includemast cell inhibitors (anti-allergics), bronchodilators, andanti-inflammatory steroids for use in the treatment of respiratorydisorders such as asthma by inhalation therapy, for example cromoglycate(e.g. the sodium salt), and albuterol (e.g. the sulfate salt). Forsystemic delivery (e.g. delivery of the bioactive agent to the systemiccirculation for the treatment of autoimmune diseases such as diabetes ormultiple sclerosis), peptides and proteins are particularly preferred.

Exemplary medicaments or bioactive agents may be selected from, forexample, analgesics, e.g. codeine, dihydromorphine, ergotamine,fentanyl, or morphine; anginal preparations, e.g. diltiazem; mast cellinhibitors, e.g. cromolyn sodium; antiinfectives, e.g. cephalosporins,macrolides, quinolines, penicillins, streptomycin, sulphonamides,tetracyclines and pentamidine; antihistamines, e.g. methapyrilene;anti-inflammatories, e.g. fluticasone propionate, beclomethasonedipropionate, flunisolide, budesonide, tripedane, cortisone, prednisone,prednisilone, dexamethasone, betamethasone, or triamcinolone acetonide;antitussives, e.g. noscapine; bronchodilators, e.g. ephedrine,adrenaline, fenoterol, formoterol, isoprenaline, metaproterenol,salbutamol, albuterol, salmeterol, terbutaline; diuretics, e.g.amiloride; anticholinergics, e.g. ipatropium, atropine, or oxitropium;lung surfactants e.g. Surfaxin, Exosurf, Survanta; xanthines, e.g.aminophylline, theophylline, caffeine; therapeutic proteins andpeptides, e.g. DNAse, insulin, glucagon, LHRH, nafarelin, goserelin,leuprolide, interferon, rhu IL-1 receptor, macrophage activation factorssuch as lymphokines and muramyl dipeptides, opioid peptides andneuropeptides such as enkaphalins, endorphins, renin inhibitors,cholecystokinins, DNAse, growth hormones, leukotriene inhibitors and thelike. In addition, bioactive agents that comprise an RNA or DNAsequence, particularly those useful for gene therapy, geneticvaccination, genetic tolerization, or antisense applications, may beincorporated in the disclosed dispersions as described herein.Representative DNA plasmids include pCMVβ (available from Genzyme Corp,Framington, Mass.) and pCMV-β-gal (a CMV promotor linked to the E. coliLac-Z gene, which codes for the enzyme β-galactosidase).

The selected bioactive agent(s) may comprise, be associated with, orincorporated in, the perforated microstructures in any form thatprovides the desired efficacy and is compatible with the chosenproduction techniques. As used herein, the terms “associate” or“associating” mean that the structural matrix or perforatedmicrostructure may comprise, incorporate, adsorb, absorb, be coated withor be formed by the bioactive agent. Where appropriate, the medicamentsmay be used in the form of salts (e.g. alkali metal or amine salts or asacid addition salts) or as esters or as solvates (hydrates). In thisregard, the form of the bioactive agents may be selected to optimize theactivity and/or stability of the medicament and/or to minimize thesolubility of the medicament in the suspension medium. It will furtherbe appreciated that, the aerosolized formulations according to theinvention may, if desired, contain a combination of two or more activeingredients. The agents may be provided in combination in a singlespecies of perforated microstructure or individually in separate speciesof perforated microstructures that are combined in the suspensionmedium. For example, two or more bioactive agents may be incorporated ina single feed stock preparation and spray dried to provide a singlemicrostructure species comprising a plurality of medicaments.Conversely, the individual medicaments could be added to separate stocksand spray dried separately to provide a plurality of microstructurespecies with different compositions. These individual species could beadded to the propellant medium in any desired proportion and placed inthe aerosol delivery system as described below. Further, as brieflymentioned above, the perforated microstructures (with or without anassociated medicament) may be combined with one or more conventionallymicronized bioactive agents to provide the desired dispersion stability.

Based on the foregoing, it will be appreciated by those skilled in theart that a wide variety of bioactive agents may be incorporated in thedisclosed stabilized dispersions. Accordingly, the list of preferredbioactive agents above is exemplary only and not intended to belimiting. It will also be appreciated by those skilled in the art thatthe proper amount of bioactive agent and the timing of the dosages maybe determined for the formulations in accordance with already existinginformation and without undue experimentation.

As seen from the passages above, various components may be associatedwith, or incorporated in the perforated microstructures of the presentinvention. Similarly, several techniques may be used to provideparticulates having the appropriate morphology (i.e. a perforatedconfiguration) and density. Among other methods, perforatedmicrostructures compatible with the instant invention may be formed bytechniques including lyophilization, spray drying, multiple emulsion,micronization, or crystallization. It will further be appreciated that,the basic concepts of many of these techniques are well known in theprior art and would not, in view of the teachings herein, require undueexperimentation to adapt them so as to provide the desired perforatedmicrostructures.

While several procedures are generally compatible with the presentinvention, particularly preferred embodiments typically compriseperforated microstructures formed by spray drying. As is well known,spray drying is a one-step process that converts a liquid feed to adried particulate form. With respect to pharmaceutical applications, itwill be appreciated that, spray drying has been used to provide powderedmaterial for various administrative routes including inhalation. See,for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols:Physical and Biological Basis for Therapy, A. J. Hickey, ed. MarcelDekkar, New York, 1996, which is incorporated herein by reference.

In general, spray drying consists of bringing together a highlydispersed liquid, and a sufficient volume of hot air to produceevaporation and drying of the liquid droplets. The preparation to bespray dried or feed (or feed stock) can be any solution, coursesuspension, slurry, colloidal dispersion, or paste that may be atomizedusing the selected spray drying apparatus. Typically, the feed issprayed into a current of warm filtered air that evaporates the solventand conveys the dried product to a collector. The spent air is thenexhausted with the solvent. Those skilled in the art will appreciatethat, several different types of apparatus may be used to provide thedesired product. For example, commercial spray dryers manufactured byBuchi Ltd. or Niro Corp. will effectively produce particles of desiredsize. It will further be appreciated that, these spray dryers, andspecifically their atomizers, may be modified or customized forspecialized applications, i.e. the simultaneous spraying of twosolutions using a double nozzle technique. More specifically, awater-in-oil emulsion can be atomized from one nozzle and a solutioncontaining an anti-adherent such as mannitol can be co-atomized from asecond nozzle. In other cases, it may be desirable to push the feedsolution though a custom designed nozzle using a high pressure liquidchromatography (HPLC) pump. Provided that microstructures comprising thecorrect morphology and/or composition are produced, the choice ofapparatus is not critical and would be apparent to the skilled artisanin view of the teachings herein.

While the resulting spray-dried powdered particles typically areapproximately spherical in shape, nearly uniform in size and frequentlyare hollow, there may be some degree of irregularity in shape dependingupon the incorporated medicament and the spray drying conditions. Inmany instances the dispersion stability of spray-dried microspheresappears to be more effective if an inflating agent (or blowing agent) isused in their production. Particularly preferred embodiments maycomprise an emulsion with the inflating agent as the disperse orcontinuous phase (the other phase being aqueous in nature). Theinflating agent is preferably dispersed with a surfactant solution,using, for instance, a commercially available microfluidizer at apressure of about 5000 to 15,000 psi. This process forms an emulsion,preferably stabilized by an incorporated surfactant, typicallycomprising submicron droplets of water immiscible blowing agentdispersed in an aqueous continuous phase. The formation of suchdispersions using this and other techniques are common and well known tothose in the art. The blowing agent is preferably a fluorinated compound(e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin,perfluorobutyl ethane) which vaporizes during the spray-drying process,leaving behind generally hollow, porous aerodynamically lightmicrospheres. As will be discussed in more detail below, other suitableblowing agents include chloroform, Freons, and hydrocarbons. Nitrogengas and carbon dioxide are also contemplated as a suitable blowingagent.

Although the perforated microstructures are preferably formed using ablowing agent as described above, it will be appreciated that, in someinstances, no blowing agent is required and an aqueous dispersion of themedicament and surfactant(s) are spray dried directly. In such cases,the formulation may be amenable to process conditions (e.g., elevatedtemperatures) that generally lead to the formation of hollow, relativelyporous microparticles. Moreover, the medicament may possess specialphysicochemical properties (e.g., high crystallinity, elevated meltingtemperature, surface activity, etc.) that make it particularly suitablefor use in such techniques.

When a blowing agent is employed, the degree of porosity of theperforated microstructure appears to depend, at least in part, on thenature of the blowing agent, its concentration in the feed stock (i.e.as an emulsion), and the spray drying conditions. With respect tocontrolling porosity, it has surprisingly been found that the use ofcompounds, heretofore unappreciated as blowing agents, may provideperforated microstructures having particularly desirablecharacteristics. More particularly, in this novel and unexpected aspectof the present invention it has been found that the use of fluorinatedcompounds having relatively high boiling points (i.e. greater than about60° C.) may be used to produce particulates that are especially suitablefor inhalation therapies. In this regard, it is possible to usefluorinated blowing agents having boiling points of greater than about70° C., 80° C., 90° C. or even 95° C. Particularly preferred blowingagents have boiling points greater than the boiling point of water, i.e.greater than 100° C. (e.g. perflubron, perfluorodecalin). In addition,blowing agents with relatively low water solubility (<⁻⁶ M) arepreferred since they enable the production of stable emulsiondispersions with mean weighted particle diameters less than 0.3 μm. Asindicated above, these blowing agents will preferably be incorporated inan emulsified feed stock prior to spray drying. For the purposes of thepresent invention this feed stock will also preferably comprise one ormore bioactive agents, one or more surfactants, or one or moreexcipients. Of course, combinations of the aforementioned components arealso within the scope of the invention

While not limiting the invention in any way it is hypothesized that, asthe aqueous feed component evaporates during spray drying it leaves athin crust at the surface of the particle. The resulting particle wallor crust formed during the initial moments of spray drying appears totrap any high boiling blowing agents as hundreds of emulsion droplets(ca. 200-300 nm). As the drying process continues, the pressure insidethe particulate increases thereby vaporizing at least part of theincorporated blowing agent and forcing it through the relatively thincrust. This venting or outgassing apparently leads to the formation ofpores or other defects in the crust. At the same time, remainingparticulate components (possibly including some blowing agent) migratefrom the interior to the surface as the particle solidifies. Thismigration apparently slows during the drying process as a result ofincreased resistance to mass transfer caused by an increased internalviscosity. Once the migration ceases, the particle solidifies, leavingvesicles, vacuoles or voids where the emulsifying agent resided. Thenumber of pores, their size, and the resulting wall thickness is largelydependent on the nature of the selected blowing agent (i.e. boilingpoint), its concentration in the emulsion, total solids concentration,and the spray-drying conditions.

It has been surprisingly found that substantial amounts of theserelatively high boiling blowing agents may be retained in the resultingspray dried product. That is, the spray dried perforated microstructuresmay comprise as much as 5%, 10%, 20%, 30% or even 40% w/w of the blowingagent. In such cases, higher production yields were obtained as a resultan increased particle density caused by residual blowing agent. It willbe appreciated by those skilled in the art that this retainedfluorinated blowing agent may alter the surface characteristics of theperforated microstructures and further increase the stability of therespiratory dispersions. Conversely, the residual blowing agent caneasily be removed with a post-production evaporation step in a vacuumoven. Optionally, pores may be formed by spray drying a bioactive agentand an excipient that can be removed from the formed microspheres undera vacuum.

In any event, typical concentrations of blowing agent in the feed stockare between 5% and 100% w/v, and more preferably, between about 20% to90% w/v. In other embodiments, blowing agent concentrations willpreferably be greater than about 10%, 20%, 30%, 40% 50% or even 60% w/v.Yet other feed stock emulsions may comprise 70%, 80%, 90% or even 95%w/v of the selected high boiling point compound.

In preferred embodiments, another method of identifying theconcentration of blowing agent used in the feed is to provide it as aratio of the concentration of the blowing agent to that of thestabilizing surfactant (i.e. phospholipid) in the precursor emulsion.For fluorocarbon blowing agents such as perfluorooctyl bromide andphosphatidylcholine, the ratio may be termed aperfluorocarbon/phosphatidylcholine ratio (or PFC/PC ratio). Whilephosphatidylcholine is used as an example, it will be appreciated thatthe appropriate surfactants may be substituted therefor. In any event,the PFC/PC ratio will range from about 1 to about 60 and morepreferably, from about 10 to about 50. For preferred embodiments, theratio will generally be greater than about 5, 10, 20, 25, 30, 40 or even50. In this respect, FIG. 1 shows a series of pictures taken ofperforated microstructures formed of phosphatidylcholine (PC) usingvarious amounts of perfluorooctyl bromide (PFC), a relatively highboiling point fluorocarbon as the blowing agent. The PFC/PC ratios areprovided under each subset of pictures, i.e. from 1A to 1F. Formationand imaging conditions are discussed in greater detail in Examples I andII below. With regard to the micrographs, the column on the left showsthe intact microstructures while the column on the right illustratescross-sections of fractured microstructures from the same preparations.

As may easily be seen in the FIG. 1, the use of higher PFC/PC ratiosprovides structures of a more hollow and porous nature. Moreparticularly, those methods employing a PFC/PC ratio of greater thanabout 4.8 tended to provide structures that are particularly compatiblewith the dispersions disclosed herein. Similarly, FIG. 2, a micrographwhich will be discussed in more detail in Example IV below, illustratesa preferably porous morphology obtained by using higher boiling pointblowing agents (in this case perfluorodecalin).

While relatively high boiling point blowing agents comprise onepreferred aspect of the instant invention, it will be appreciated thatmore conventional blowing or inflating agents may also be used toprovide compatible perforated microstructures. Generally, the inflatingagent can be any material that will turn to a gas at some point duringthe spray drying or post-production process. Suitable agents include:

1. Dissolved low-boiling (below 100° C.) solvents with limitedmiscibility with aqueous solutions, such as methylene chloride, acetoneand carbon disulfide used to saturate the solution at room temperature.

2. A gas, e.g. CO₂ or N₂, used to saturate the solution at roomtemperature and elevated pressure (e.g. 3 bar). The droplets are thensupersaturated with the gas at 1 atmosphere and 100° C.

3. Emulsions of immiscible low-boiling (below 100° C.) liquids such asFreon 113, perfluoropentane, perfluorohexane, perfluorobutane, pentane,butane, FC-11, FC-11B1, FC-11B2, FC-12B2, FC-21, FC-21B1, FC-21B2,FC-31B1, FC-113A, FC-122, FC-123, FC-132, FC-133, FC-141, FC-141B,FC-142, FC-151, FC-152, FC-1112, FC-1121 and FC-1131.

With respect to these lower boiling point inflating agents, they aretypically added to the feed stock in quantities of about 1% to 80% w/vof the surfactant solution. Approximately 30% w/v inflating agent hasbeen found to produce a spray dried powder that may be used to form thestabilized dispersions of the present invention.

Regardless of which blowing agent is ultimately selected, it has beenfound that compatible perforated microstructures may be producedparticularly efficiently using a Büchi mini spray drier (model B-191,Switzerland). As will be appreciated by those skilled in the art, theinlet temperature and the outlet temperature of the spray drier are notcritical but will be of such a level to provide the desired particlesize and to result in a product that has the desired activity of themedicament. In this regard, the inlet and outlet temperatures areadjusted depending on the melting characteristics of the formulationcomponents and the composition of the feed stock. The inlet temperaturemay thus be between 60° C. and 170° C., with the outlet temperatures ofabout 40° C. to 120° C. depending on the composition of the feed and thedesired particulate characteristics. Preferably, these temperatures willbe from 90° C. to 120° C. for the inlet and from 60° C. to 90° C. forthe outlet. The flow rate which is used in the spray drying equipmentwill generally be about 3 ml per minute to about 15 ml per minute. Theatomizer air flow rate will vary between values of 1,200 liters per hourto about 3,900 liters per hour. Commercially available spray dryers arewell known to those in the art, and suitable settings for any particulardispersion can be readily determined through standard empirical testing,with due reference to the examples that follow. Of course, theconditions may be adjusted so as to preserve biological activity inlarger molecules such as proteins or peptides.

Particularly preferred embodiments of the present invention comprisespray drying preparations comprising a surfactant such as a phospholipidand at least one bioactive agent. In other embodiments, the spray dryingpreparation may further comprise an excipient comprising a hydrophilicmoiety such as, for example, a carbohydrate (i.e. glucose, lactose, orstarch) in addition to any selected surfactant. In this regard, variousstarches and derivatized starches suitable for use in the presentinvention. Other optional components may include conventional viscositymodifiers, buffers such as phosphate buffers or other conventionalbiocompatible buffers or pH adjusting agents such as acids or bases, andosmotic agents (to provide isotonicity, hyperosmolarity, orhyposmolarity). Examples of suitable salts include sodium phosphate(both monobasic and dibasic), sodium chloride, calcium phosphate,calcium chloride and other physiologically acceptable salts.

Whatever components are selected, the first step in particulateproduction typically comprises feed stock preparation. Preferably, theselected drug is dissolved in water to produce a concentrated solution.The drug may also be dispersed directly in the emulsion, particularly inthe case of water insoluble agents. Alternatively, the drug may beincorporated in the form of a solid particulate dispersion. Theconcentration of the drug used is dependent on the dose of drug requiredin the final powder and the performance of the MDI drug suspension(e.g., fine particle dose). As needed, co-surfactants such as poloxamer188 or span 80 may be added to this annex solution. Additionally,excipients such as sugars and starches can also be added.

In selected embodiments an oil-in-water emulsion is then formed in aseparate vessel. The oil employed is preferably a fluorocarbon (e.g.,perfluorooctyl bromide, perfluorodecalin) which is emulsified using asurfactant such as a long chain saturated phospholipid. For example, onegram of phospholipid may be homogenized in 150 g hot distilled water(e.g., 60° C.) using a suitable high shear mechanical mixer (e.g.,Ultra-Turrax model T-25 mixer) at 8000 rpm for 2 to 5 minutes. Typically5 to 25 g of fluorocarbon is added dropwise to the dispersed surfactantsolution while mixing. The resulting perfluorocarbon in water emulsionis then processed using a high pressure homogenizer to reduce theparticle size. Typically the emulsion is processed at 12,000 to 18,000psi, 5 discrete passes and kept at 50 to 80° C.

The drug solution and perfluorocarbon emulsion are then combined and fedinto the spray dryer. Typically the two preparations will be miscible asthe emulsion will preferably comprise an aqueous continuous phase. Whilethe bioactive agent is solubilized separately for the purposes of theinstant discussion it will be appreciated that, in other embodiments,the bioactive agent may be solubilized (or dispersed) directly in theemulsion. In such cases, the bioactive emulsion is simply spray driedwithout combining a separate drug preparation.

In any event, operating conditions such as inlet and outlet temperature,feed rate, atomization pressure, flow rate of the drying air, and nozzleconfiguration can be adjusted in accordance with the manufacturer'sguidelines in order to produce the required particle size, andproduction yield of the resulting dry microstructures. Exemplarysettings are as follows: an air inlet temperature between 60° C. and170° C.; an air outlet between 40° C. to 120° C.; a feed rate between 3ml to about 15 ml per minute; and an aspiration setting of 100% and anatomization air flow rate between 1,200 to 2,800 L/hr. The selection ofappropriate apparatus and processing conditions are well within thepurview of a skilled artisan in view of the teachings herein and may beaccomplished without undue experimentation. It will be appreciated that,the use of these and substantially equivalent methods provide for theformation of hollow porous aerodynamically light microspheres withparticle diameters appropriate for aerosol deposition into the lung.

Along with spray drying the perforated microstructures of the presentinvention may be formed by lyophilization. Those skilled in the art willappreciate that, lyophilization is a freeze-drying process in whichwater is sublimed from the composition after it is frozen. Theparticular advantage associated with the lyophilization process is thatbiologicals and pharmaceuticals that are relatively unstable in anaqueous solution can be dried without elevated temperatures (therebyeliminating the adverse thermal effects), and then stored in a dry statewhere there are few stability problems. With respect to the instantinvention such techniques are particularly compatible with theincorporation of peptides, proteins, genetic material and other naturaland synthetic macromolecules in the perforated microstructures withoutcompromising physiological activity. Methods for providing lyophilizedparticulates are known to those of skill in the art and it would clearlynot require undue experimentation to provide dispersion compatiblemicrostructures in accordance with the teachings herein. Accordingly, tothe extent that lyophilization processes may be used to providemicrostructures having the desired porosity and size they areconformance with the teachings herein and are expressly contemplated asbeing within the scope of the instant invention.

In addition to the aforementioned techniques, the perforatedmicrostructures of the present invention may also be formed using adouble emulsion method. In the double emulsion method the medicament isfirst dispersed in a polymer dissolved in an organic solvent (e.g.methylene chloride) by sonication or homogenization. This primaryemulsion is then stabilized by forming a multiple emulsion in acontinuous aqueous phase containing an emulsifier such aspolyvinylalcohol. The organic solvent is then removed by evaporation orextraction using conventional techniques and apparatus. The resultingmicrospheres are washed, filtered and dried prior to combining them withan appropriate suspension medium in accordance with the presentinvention.

As extensively discussed above, the stabilized dispersions of thepresent invention further comprise a continuous phase suspension medium.It is an advantage of the present invention that any biocompatiblesuspension medium having adequate vapor pressure to act as a propellantmay be used. Particularly preferred suspension media are compatible withuse in a metered dose inhaler. That is, they will be able to formaerosols upon the activation of the metering valve and associatedrelease of pressure. In general, the selected suspension medium shouldbe biocompatible (i.e. relatively non-toxic) and non-reactive withrespect to the suspended perforated microstructures comprising thebioactive agent. Preferably, the suspension medium will not act as asubstantial solvent for any components incorporated in the perforatedmicrospheres. Selected embodiments of the invention comprise suspensionmedia selected from the group consisting of fluorocarbons (includingthose substituted with other halogens), hydrofluoroalkanes,perfluorocarbons, hydrocarbons, alcohols, ethers or combinations thereofIt will be appreciated that, the suspension medium may comprise amixture of various compounds selected to impart specificcharacteristics.

Particularly suitable propellants for use in the suspension mediums ofthe present invention are those propellant gases that can be liquefiedunder pressure at room temperature and, upon inhalation or topical use,are safe, toxicologically innocuous and free of side effects. In thisregard, compatible propellants may comprise any hydrocarbon,fluorocarbon, hydrogen-containing fluorocarbon or mixtures thereofhaving a sufficient vapor pressure to efficiently form aerosols uponactivation of a metered dose inhaler. Those propellants typically termedhydrofluoroalkanes or HFAs are especially compatible. Suitablepropellants include, for example, short chain hydrocarbons, C₁₋₄hydrogen-containing chlorofluorocarbons such as CH₂ClF, CCl₂F₂CHClF,CF₃CHClF, CHF₂CClF₂, CHClFCHF₂, CF₃CH₂Cl, and CClF₂CH₃; C₁₋₄hydrogen-containing fluorocarbons (e.g. HFAs) such as CHF₂CHF₂, CF₃CH₂F,CHF₂CH₃, and CF₃CHFCF₃; and perfluorocarbons such as CF₃CF₃ andCF₃CF₂CF₃. Preferably, a single perfluorocarbon or hydrogen-containingfluorocarbon is employed as the propellant. Particularly preferred aspropellants are 1,1,1,2-tetrafluoroethane (CF₃CH₂F) (HFA-134a) and1,1,1,2,3,3,3-heptafluoro-n-propane (CF₃CHFCF₃) (HFA-227),perfluoroethane, monochlorodifluoromethane, 1,1-difluoroethane, andcombinations thereof. It is desirable that the formulations contain nocomponents that deplete stratospheric ozone. In particular it isdesirable that the formulations are substantially free ofchlorofluorocarbons such as CCl₃F, CCl₂F₂, and CF₃CCl₃.

Specific fluorocarbons, or classes of fluorinated compounds, that areuseful in the suspension media include, but are not limited to,fluoroheptane, fluorocycloheptane, fluoromethylcycloheptane,fluorohexane, fluorocyclohexane, fluoropentane, fluorocyclopentane,fluoromethylcyclopentane, fluorodimethylcyclopentanes,fluoromethylcyclobutane, fluorodimethylcyclobutane,fluorotrimethylcyclobutane, fluorobutane, fluorocyclobutane,fluoropropane, fluoroethers, fluoropolyethers and fluorotriethylamines.It will be appreciated that, these compounds may be used alone or incombination with more volatile propellants. It is a distinct advantagethat such compounds are generally environmentally sound and biologicallynon-reactive.

In addition to the aforementioned fluorocarbons and hydrofluoroalkanes,various chlorofluorocarbons and substituted fluorinated compounds mayalso be used as suspension mediums in accordance with the teachingsherein. In this respect, FC-11 (CCL3F), FC-11B1 (CBrCl2F), FC-11B2(CBr2ClF), FC12B2 (CF2Br2), FC21 (CHC12F), FC21B1 (CHBrClF), FC-21B2(CHBr2F), FC-31B1 (CH2BrF), FC113A (CC13CF3), FC-122 (CClF2CHC12, FC-123(CF3CHC12), FC-132 (CHClFCHClF), FC-133 (CHClFCHF2), FC-141(CH2ClCHClF), FC-141B (CCl2FCH3), FC-142 (CHF2CH2Cl), FC-151(CH2FCH2Cl), FC-152 (CH2FCH2F), FC-1112 (CClF=CClF), FC-1121 (CHCl=CFCl)and FC-1131 (CHCl=CHF) are all compatible with the teachings hereindespite possible attendant environmental concerns. As such, each ofthese compounds may be used, alone or in combination with othercompounds (i.e. less volatile fluorocarbons) to form the stabilizedrespiratory dispersions of the present invention.

With respect to possible media combinations, relatively volatilecompounds may be mixed with lower vapor pressure components to providesuspension media having specified physical characteristics selected toflirter improve stability or enhance the bioavailability of thedispersed bioactive agent. In preferred embodiments, the lower vaporpressure compounds will comprise fluorinated compounds (e.g.fluorocarbons) having a boiling point greater than about 25° C.Particularly preferred lower vapor pressure fluorinated compounds foruse in the suspension medium may comprise of perfluorooctylbromideC₈F₁₇Br (PFOB or perflubron), dichlorofluorooctane C₈F₁₆Cl₂,perfluorooctylethane C₈F₁₇C₂H₅ (PFOE), perfluorodecylbromide C₁₀F₂₁,Br(PFDB) or perfluorobutylethane C₄F₉C₂H₅. Preferably, these lower vaporpressure compounds are present in a relatively low level. Such compoundsmay be added directly to the suspension medium or may be associated withthe perforated microstructures.

Similarly, as indicated above, it is an advantage of the presentinvention that stabilized dispersions may be formed in HFA or PFCpropellants without the use of additional cosolvents or adjuvants.Accordingly, in selected embodiments the formulations are substantiallyfree of potentially reactive liquid components of higher polarity thanthe propellant employed. This is largely because the presence ofcosolvents or adjuvants could potentially increase the solubility of theperforated particles in the suspension medium, thereby altering particlemorphology, and particle size (growth by Ostwald ripening) over time.However, depending on the perforated microstructure composition, or theselection of propellant, it may be desirable to include an appropriatecosolvent or adjuvant to adjust vapor pressure or increaseadministration efficiency. As such, it is expressly contemplated that anHFA propellant containing suspension medium may additionally contain anadjuvant or cosolvent as long as it does not adversely impact thestability of the particles. For example propane, ethanol, isopropylalcohol, butane, isobutane, pentane, isopentane or a dialkyl ether suchas dimethyl ether may be incorporated in the suspension media.Similarly, the suspension medium may contain a volatile fluorocarbon. Ingeneral, up to 50% w/w of the propellant may comprise a volatileadjuvant such as a hydrocarbon or fluorocarbon. More preferably, thesuspension medium will comprise less than about 40%, 30%, 20% or 10% w/wof cosolvent or adjuvant.

It will further be appreciated that, one of ordinary skill in the artcan readily determine other compounds that would perform suitably in thepresent invention which apparently do not exhibit a desirable vaporpressure and/or viscosity. Rather, it will be understood that, certaincompounds outside the preferred ranges of vapor pressure or viscositycan be used if they provide the desired aerosolized medicament uponactivation of a MDI.

The stabilized suspensions or dispersions of the present invention maybe prepared by dispersal of the microstructures in the selectedsuspension medium which may then be placed in a container or reservoir.In this regard, the stabilized preparations of the present invention canbe made by simply combining the components in sufficient quantity toproduce the final desired dispersion concentration. Although themicrostructures readily disperse without mechanical energy, theapplication of energy (e.g., sonication or stirring) to aid indispersion is expressly contemplated as being within the scope of theinvention. Alternatively, the components may be mixed by simple shakingor other type of agitation. The process is preferably carried out underanhydrous conditions to obviate any adverse effects of moisture onsuspension stability. Once formed, the dispersion has a reducedsusceptibility to flocculation and sedimentation.

The remarkable stability provided by the preparations of the instantinvention is graphically illustrated in FIGS. 3A to 3D where a MDIformulation prepared in accordance with the present invention (as willbe discussed more fully in Example XVIII below) is compared with acommercially available MDI formulation. In each of the pictures, takenat 0 seconds, 30 seconds, 60 seconds and 2 hours after shaking, thecommercial formulation is on the left, and the perforated microstructuredispersion formed accordance with the present invention is on the right.Whereas the commercial cromolyn sodium formulation shows creaming within30 seconds of mixing, almost no creaming is noted in the spray-driedparticles after as long as 2 hours. Moreover, there was little creamingin perforated microstructure formulation after 4 hours (not shown). Thisexample clearly illustrates the stability that can be achieved when thehollow porous particles of compatible materials are filled with thesuspension medium (i.e. in the form of a homodispersion).

It will also be understood that, other components can be included in thepharmaceutical compositions of the present invention. For example,osmotic agents, stabilizers, chelators, buffers, hygroscopic agents,viscosity modulators, salts, and sugars can be added to fine tune thestabilized dispersions for maximum life and ease of administration. Suchcomponents may be added directly to the suspension medium or associatedwith, or incorporated in, the dispersed perforated microstructures.Considerations such as sterility, isotonicity, and biocompatibility maygovern the use of conventional additives to the disclosed compositions.The use of such agents will be understood to those of ordinary skill inthe art and the specific quantities, ratios, and types of agents can bedetermined empirically without undue experimentation.

Conventional bulk manufacturing methods and machinery well known tothose skilled in the art of pharmaceutical manufacture may be employedfor the preparation of large scale batches for commercial production offilled canisters, or reservoirs for MDIs. With MDIs for example, in onebulk manufacturing method, a metering valve is crimped onto an aluminumcan to provide an empty canister or reservoir. The perforatedmicroparticles are added to a charge vessel, and a liquefied propellant(suspension medium) is pressure-filled through the charge vessel into amanufacturing vessel. The respiratory blend or drug suspension is mixedbefore recirculation to a filling machine and an aliquot of thestabilized dispersion is then filled through the metering valve into thereservoir. Typically, in batches prepared for pharmaceutical use, eachfilled canister is check-weighed, coded with a batch number and packedinto a tray for storage before release testing.

In other embodiments, the perforated microparticles are introduced intoan empty reservoir which is then crimp-sealed to the metering valve. Thereservoir or canister is then charged with HFA propellant by overpressure through the valve stem. In yet another embodiment, thestabilized dispersion may be prepared outside the canister or reservoirand then introduced cold filling techniques. The canister is thencrimped sealed. Those skilled in the art will appreciated that thefilling procedure selected will, at least to some extent, depend on thetype of valve chosen.

The canisters generally comprise a container or reservoir capable ofwithsanding the vapor pressure of the propellant used such as, a plasticor plastic-coated glass bottle, or preferably, a metal can or, forexample, an aluminum can which may optionally be anodized,lacquer-coated and/or plastic-coated, wherein the container is closedwith a metering valve. The metering valves are designed to deliver ametered amount of the formulation per actuation. The valves incorporatea gasket to prevent leakage of propellant through the valve. The gasketmay comprise any suitable elastomeric material such as, for example, lowdensity polyethylene, chlorobutyl, black and whitebutadiene-acrylonitrile rubbers, butyl rubber and neoprene. Suitablevalves are commercially available from manufacturers well known in theaerosol industry, for example, from Valois, France (e.g. DFIO, DF30, DF31/50 ACT, DF60), Bespak plc, LIK (e.g. BK300, BK356) and 3M-NeotechnicLtd., LIK (e.g. Spraymiser).

Each filled canister is conveniently fitted into a suitable channelingdevice prior to use to form a metered dose inhaler for administration ofthe medicament into the lungs or nasal cavity of a patient. Suitablechanneling devices comprise for example a valve actuator and acylindrical or cone-like passage through which medicament may bedelivered from the filled canister via the metering valve, to the noseor mouth of a patient e.g., a mouthpiece actuator. Metered dose inhalersare designed to deliver a fixed unit dosage of medicament per actuationsuch as, for example, in the range of 10 to 5000 micrograms of bioactiveagent per actuation. Typically, a single charged canister will providefor tens or even hundreds of shots or doses.

It will be appreciated that, the stabilized preparations for use inmetered dose inhalers of the present invention may be advantageouslysupplied to the physician or other health care professional, in asterile, prepackaged or kit form. More particularly, the formulationsmay be supplied as charged MDI reservoirs or canisters, ready foradministration. Such kits may contain a number of charged canisters,preferably along with a disposable actuator. In this regard, the patientmay then change or substitute canisters during a particular course oftreatment. It will also be appreciated that, such kits may include asingle charged canister associated or affixed to an actuator, or thatthe preparation may be supplied in a disposable MDI device.

Administration of bioactive agent may be indicated for the treatment ofmild, moderate or severe, acute or chronic symptoms or for prophylactictreatment. Moreover, the bioactive agent may be administered to treatlocal or systemic conditions or disorders. It will be appreciated that,the precise dose administered will depend on the age and condition ofthe patient, the particular medicament used and the frequency ofadministration and will ultimately be at the discretion of the attendantphysician. When combinations of bioactive agents are employed, the doseof each component of the combination will generally be the same as thatemployed for each component when used alone.

As discussed throughout the specification, the stabilized dispersionsdisclosed herein are preferably administered to the lung or pulmonaryair passages of a patient via aerosolization, such as with a metereddose inhaler. MDIs are well known in the art and could easily beemployed for administration of the claimed dispersions without undueexperimentation. Breath activated MDIs, as well as those comprisingother types of improvements which have been, or will be, developed arealso compatible with the stabilized dispersions and present inventionand, as such, are contemplated as being with in the scope thereofHowever, it should be emphasized that, in preferred embodiments, thestabilized dispersions may be administered using a number of differentroutes including, but not limited to, topical, nasal, pulmonary or oral.Those skilled in the art will appreciate that, such routes are wellknown and that the dosing and administration procedures may be easilyderived for the stabilized dispersions of the present invention.

More efficient delivery of the aerosolized medicament to the bronchialairways has several important clinical implications. Among suchadvantages are: reduced cost of diagnosis and therapy due to reductionin the amount of aerosolized material required to generate a clinicalresult; smaller, more effective and more efficient patient dosing at thedesired site (i.e., the lung or bronchus); and reduced side effects dueto less deposition in the throat. Such advantages may in turn help toincrease overall patient compliance.

The foregoing description will be more fully understood with referenceto the following Examples. Such Examples, are, however, merelyrepresentative of preferred methods of practicing the present inventionand should not be read as limiting the scope of the invention.

I Preparation of Hollow Porous Particles of Gentamicin Sulfate bySpray-Drying

40 to 60 ml of the following solutions were prepared for spray drying:

50% w/w hydrogenated phosphatidylcholine, E-100-3 (Lipoid KG,Ludwigshafen, Germany)

50% w/w gentamicin sulfate (Amresco, Solon, Ohio)

Perfluorooctylbromide, Perflubron (NMK, Japan)

Deionized water

Perforated microstructures comprising gentamicin sulfate were preparedby a spray drying technique using a B-191 Mini Spray-Drier (Büchi,Flawil, Switzerland) under the following conditions: aspiration: 100%,inlet temperature: 85° C.; outlet temperature: 61° C.; feed pump: 10%;N₂ flow: 2,800 L/hr. Variations in powder porosity were examined as afunction of the blowing agent concentration.

Fluorocarbon-in-water emulsions of perfluorooctyl bromide containing a1:1 w/w ratio of phosphatidylcholine (PC), and gentamicin sulfate wereprepared varying only the PFC/PC ratio. Hydrogenated eggphosphatidylcholine (1.3 grams) was dispersed in 25 mL deionized waterusing an Ultra-Turrax mixer (model T-25) at 8000 rpm for 2 to 5 minutes(T=60-70° C.). A range from 0 to 40 grams of perflubron was addeddropwise during mixing (T=60-70° C.). After addition was complete, thefluorocarbon-in-water emulsion was mixed for an additional period of notless than 4 minutes. The resulting coarse emulsions were thenhomogenized under high pressure with an Avestin (Ottawa, Canada)homogenizer at 15,000 psi for 5 passes. Gentamicin sulfate was dissolvedin approximately 4 to 5 mL deionized water and subsequently mixed withthe perflubron emulsion immediately prior to the spray dry process. Thegentamicin powders were then obtained by spray drying using theconditions described above. A free flowing, pale yellow powder wasobtained from all perflubron containing emulsions. The yield for each ofthe various formulations ranged from 35% to 60%.

II Morphology of Gentamicin Sulfate Spray-Dried Powders

A strong dependence of the powder morphology, degree of porosity, andproduction yield was observed as a function of the PFC/PC ratio byscanning electron microscopy (SEM). A series of six SEM micrographsillustrating these observations, labeled 1A1 to 1F1, are shown in theleft hand column of FIG. 1. As seen in these micrographs the porosityand surface roughness was found to be highly dependent on theconcentration of the blowing agent, where the surface roughness, numberand size of the pores increased with increasing PFC/PC ratios. Forexample, the formulation devoid of perfluorooctyl bromide producedmicrostructures that appeared to be highly agglomerated and readilyadhered to the surface of the glass vial. Similarly, smooth, sphericallyshaped microparticles were obtained when relatively little (PFC/PCratio=1.1 or 2.2) blowing agent was used. As the PFC/PC ratio wasincreased the porosity and surface roughness increased dramatically.

As shown in the right hand column of FIG. 1, the hollow nature of themicrostructures was also enhanced by the incorporation of additionalblowing agent. More particularly, the series of six micrographs labeled1A2 to 1F2 show cross sections of fractured microstructures as revealedby transmission electron microscopy (TEM). Each of these images wasproduced using the same microstructure preparation as was used toproduce the corresponding SEM micrograph in the left hand column. Boththe hollow nature and wall thickness of the resulting perforatedmicrostructures appeared to be largely dependent on the concentration ofthe selected blowing agent. That is, the hollow nature of thepreparation appeared to increase and the thickness of the particle wallsappeared to decrease as the PFC/PC ratio increased. As may be seen inFIGS. 1A2 to 1C2 substantially solid structures were obtained fromformulations containing little or no fluorocarbon blowing agent.Conversely, the perforated microstructures produced using a relativelyhigh PFC /PC ratio of approximately 45 (shown in FIG. 1F2 proved to beextremely hollow with a relatively thin wall ranging from about 43.5 to261nm.

III Preparation of Hollow Porous Particles of Albuterol Sulfate bySpray-Drying

Hollow porous albuterol sulfate particles were prepared by aspray-drying technique with a B-191 Mini Spray-Drier (Büchi, Flawil,Switzerland) under the following spray conditions: aspiration: 100%,inlet temperature: 85° C.; outlet temperature: 61° C.; feed pump: 10%;N₂ flow: 2,800 L/hr. The feed solution was prepared by mixing twosolutions A and B immediately prior to spray drying.

Solution A: 20 g of water was used to dissolve 1 g of albuterol sulfate(Accurate Chemical, Westbury, N.Y.) and 0.021 g of poloxamer 188 NFgrade (BASF, Mount Olive, N.J.).

Solution B: A fluorocarbon-in-water emulsion stabilized by phospholipidwas prepared in the following manner. The phospholipid, 1 g EPC-100-3(Lipoid KG, Ludwigshafen, Germany), was homogenized in 150 g of hotdeionized water (T=50 to 60° C.) using an Ultra-Turrax mixer (modelT-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). 25 g ofperfluorooctyl bromide (Atochem, Paris, France) was added dropwiseduring mixing. After the fluorocarbon was added, the emulsion was mixedfor a period of not less than 4 minutes. The resulting coarse emulsionwas then passed through a high pressure homogenizer (Avestin, Ottawa,Canada) at 18,000 psi for 5 passes.

Solutions A and B were combined and fed into the spray-dryer under theconditions described above. A free flowing, white powder was collectedat the cyclone separator. The hollow porous albuterol sulfate particleshad a volume-weighted mean aerodynamic diameter of 1.18±1.42 μm asdetermined by a time-of-flight analytical method (Aerosizer, AmherstProcess Instruments, Amherst, Mass.). Scanning electron microscopy (SEM)analysis showed the powders to be spherical and highly porous. The tapdensity of the powder was determined to be less than 0.1 g/cm³.

This foregoing example serves to illustrate the inherent diversity ofthe present invention as a drug delivery platform capable of effectivelyincorporating any one of a number of pharmaceutical agents. Theprinciple is further illustrated in the next example.

IV

Preparation of Hollow Porous Particles of Cromolyn Sodium bySpray-Drying

Perforated microstructures comprising cromolyn sodium were prepared by aspray-drying drying technique with a B-191 Mini Spray-Drier (Büchi,Flawil, Switzerland) under the following spray conditions: aspiration:100%, inlet temperature: 85° C.; outlet temperature: 61° C.; feed pump:10%; N₂ flow: 2,800 L/hr. The feed solution was prepared by mixing twosolutions A and B immediately prior to spray drying.

Solution A: 20 g of water was used to dissolve 1 g of cromolyn sodium(Sigma Chemical Co, St. Louis, Mo.) and 0.021 g of poloxamer 188 NFgrade (BASF, Mount Olive, N.J.).

Solution B: A fluorocarbon-in-water emulsion stabilized by phospholipidwas prepared in the following manner. The phospholipid, 1 g EPC-100-3(Lipoid KG, Ludwigshafen, Germany), was homogenized in 150 g of hotdeionized water (T=50 to 60° C.) using an Ultra-Turrax mixer (modelT-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). 27 g ofperfluorodecalin (Air Products, Allentown, Pa.) was added dropwiseduring mixing. After the fluorocarbon was added, the emulsion was mixedfor at least 4 minutes. The resulting coarse emulsion was then passedthrough a high pressure homogenizer (Avestin, Ottawa, Canada) at 18,000psi for 5 passes.

Solutions A and B were combined and fed into the spray dryer under theconditions described above. A free flowing, pale yellow powder wascollected at the cyclone separator. The hollow porous cromolyn sodiumparticles had a volume-weighted mean aerodynamic diameter of 1.23±1.31μm as determined by a time-of-flight analytical method (Aerosizer,Amherst Process Instruments, Amherst, Mass.) As shown in FIG. 2,scanning electron microscopy (SEM) analysis showed the powders to beboth hollow and porous. The tap density of the powder was determined tobe less than 0.1 g/cm³.

V Preparation of Hollow Porous Particles of BDP by Spray-Drying

Perforated microstructures comprising beclomethasone dipropionate (BDP)particles were prepared by a spray-drying technique with a B-191 MiniSpray-Drier (Büchi, Flawil, Switzerland) under the following sprayconditions: aspiration: 100%, inlet temperature: 85° C.; outlettemperature: 61° C.; feed pump: 10%; N₂ flow: 2,800 L/hr. The feed stockwas prepared by mixing 0.11 g of lactose with a fluorocarbon-in-wateremulsion immediately prior to spray drying. The emulsion was prepared bythe technique described below.

74 mg of BDP (Sigma, Chemical Co., St. Louis, Mo.), 0.5 g of EPC-100-3(Lipoid KG, Ludwigshafen, Germany), 15mg sodium oleate (Sigma), and 7 mgof poloxamer 188 (BASF, Mount Olive, N.J.) were dissolved in 2 ml of hotmethanol. The methanol was then evaporated to obtain a thin film of thephospholipid/steroid mixture. The phospholipid/steroid mixture was thendispersed in 64 g of hot deionized water (T=50 to 60° C.) using anUltra-Turrax mixer (model T-25) at 8000 rpm for 2 to 5 minutes (T=60-70°C.). 8 g of perflubron (Atochem, Paris, France) was added dropwiseduring mixing. After the addition was complete, the emulsion was mixedfor an additional period of not less than 4 minutes. The resultingcoarse emulsion was then passed through a high pressure homogenizer(Avesthn, Ottawa, Canada) at 18,000 psi for 5 passes. This emulsion wasthen used to form the feed stock which was spray dried as describedabove. A free flowing, white powder was collected at the cycloneseparator. The hollow porous BDP particles had a tap density of lessthan 0.1 g/cm³.

VI Preparation of Hollow Porous Particles of TAA by Spray-Drying

Perforated microstructures comprising triamcinolone acetonide (TAA)particles were prepared by a spray drying technique with a B-191 MiniSpray-Drier (Büchi, Flawil, Switzerland) under the following sprayconditions: aspiration: 100%, inlet temperature: 85° C.; outlettemperature: 61° C.; feed pump: 10%; N₂ flow: 2,800 Lthr. The feed stockwas prepared by mixing 0.57 g of lactose with a fluorocarbon-in-wateremulsion immediately prior to spray drying. The emulsion was prepared bythe technique described below.

100 mg of TAA (Sigma, Chemical Co., St. Louis, Mo.), 0.56 g of EPC-100-3(Lipoid KG, Ludwigshafen, Germany), 25mg sodium oleate (Sigma), and 13mg of poloxamer 188 (BASF, Mount Olive, N.J.) were dissolved in 2 ml ofhot methanol. The methanol was then evaporated to obtain a thin film ofthe phospholipid/steroid mixture. The phospholipid/steroid mixture wasthen dispersed in 64 g of hot deionized water (T=50 to 60° C.) using anUltra-Turrax mixer (model T-25) at 8000 rpm for 2 to 5 minutes (T=60-70°C.). 8 g of perflubron (Atochem, Paris, France) was added dropwiseduring mixing. After the fluorocarbon was added, the emulsion was mixedfor at least 4 minutes. The resulting coarse emulsion was then passedthrough a high pressure homogenizer (Avestin, Ottawa, Canada) at 18,000psi for 5 passes. This emulsion was then used to form the feed stockwhich was spray dried as described above. A free flowing, white powderwas collected at the cyclone separator. The hollow porous TAA particleshad a tap density of less than 0.1 g/cm³.

VII Preparation of Hollow Porous Particles of DNase I by Spray-Drying

Hollow porous DNase I particles were prepared by a spray dryingtechnique with a B-191 Mini Spray-Drier (Büchi, Flawil, Switzerland)under the following conditions: aspiration: 100%, inlet temperature: 80°C.; outlet temperature: 61° C.; feed pump: 10%; N₂ flow: 2,800 L/hr. Thefeed was prepared by mixing two solutions A and B immediately prior tospray drying.

Solution A: 20 g of water was used to dissolve 0.5 gr of human pancreasDNase I (Calbiochem, San Diego Calif.) and 0.012 g of poloxamer 188 NFgrade (BASF, Mount Olive, N.J.).

Solution B: A fluorocarbon-in-water emulsion stabilized by phospholipidwas prepared in the following way. The phospholipid, 0.52 g EPC-100-3(Lipoid KG, Ludwigshafen, Germany), was homogenized in 87 g of hotdeionized water (T=50 to 60° C.) using an Ultra-Turrax mixer (modelT-25) at 8000 rpm for 2 to 5 minutes (T=60-70° C.). 13 g of perflubron(Atochem, Paris, France) was added dropwise during mixing. After thefluorocarbon was added, the emulsion was mixed for at least 4 minutes.The resulting coarse emulsion was then passed through a high pressurehomogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes.

Solutions A and B were combined and fed into the spray dryer under theconditions described above. A free flowing, pale yellow powder wascollected at the cyclone separator. The hollow porous DNase I particleshad a volume-weighted mean aerodynamic diameter of 1.29±1.40 μm asdetermined by a time-of-flight analytical method (Aerosizer, AmherstProcess Instruments, Amherst, Mass.). Scanning electron microscopy (SEM)analysis showed the powders to be both hollow and porous. The tapdensity of the powder was determined to be less than 0.1 g/cm³.

The foregoing example further illustrates the extraordinarycompatibility of the present invention with a variety of bioactiveagents. That is, in addition to relatively small, hardy compounds suchas steroids, the preparations of the present invention may be formulatedto effectively incorporate larger, fragile molecules such as proteinsand genetic material.

VIII Preparation of hollow porous powder by spray drying a gas-in-wateremulsion

The following solutions were prepared with water for injection:

Solution 1:

Solution 1  3.9% w/v m-HES hydroxyethylstarch (Ajinomoto, Tokyo, Japan)3.25% w/v Sodium chloride (Mallinckrodt, St. Louis, MO) 2.83% w/v Sodiumphosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.42% w/v Sodiumphosphate, monobasic (Mallinckrodt, St. Louis, MO) Solution 2 0.45% w/vPoloxamer 188 (BASF, Mount Olive, NJ) 1.35% w/v Hydrogenated eggphosphatidylcholine, EPC-3 (Lipoid KG, Ludwigshafen, Germany)

The ingredients of solution 1 were dissolved in warm water using a stirplate. The surfactants in solution 2 were dispersed in water using ahigh shear mixer. The solutions were combined following emulsificationand saturated with nitrogen prior to spray drying.

The resulting dry, free flowing, hollow spherical product had a meanparticle diameter of 2.6±1.5 μm. The particles were spherical and porousas determined by SEM.

The previous example illustrates the point that a wide variety ofblowing agents (here nitrogen) may be used to provide microstructuresexhibiting the desired morphology. Indeed, one of the primary advantagesof the present invention is the ability to alter formation conditions soas to preserve biological activity (i.e. with proteins), or to producemicrostructures having selected porosity.

IX Preparation of Metered Dose Inhalers Containing Hollow PorousParticles

A pre-weighed amount of the hollow porous particles prepared in ExamplesI, III, IV, V, VI, and VII were placed into 10 ml aluminum cans, anddried in a vacuum oven under the flow of nitrogen for 3-4 hours at 40°C. The amount of powder filled into the can was determined by the amountof drug required for therapeutic effect. After this, the can was crimpsealed using a DF31/50 act 50 μl valve (Valois of America, Greenwich,Conn.) and filled with HFA-134a (DuPont, Wilmington, Del.) propellant byoverpressure through the stem. The amount of the propellant in the canwas determined by weighing the can before and after the fill.

X Andersen Impactor Test for Assessing MDI Performance

The MDIs prepared in Example IX were then tested using commonly acceptedpharmaceutical procedures. The method utilized was compliant with theU.S. Pharmacopeia (USP) procedure (Pharmacopeial Previews (1996)22:3065-3098) incorporated herein by reference. After 5 shots to waste,20 shots from the test MDI were made into an Andersen Impactor.

Extraction procedure. The extraction from all the plates, inductionport, and actuator were performed in closed vials with 10 mnL of asuitable solvent. The filter was installed but not assayed, because thepolyacrylic binder interfered with the analysis. The mass balance andparticle size distribution trends indicated that the deposition on thefilter was negligibly small. Methanol was used for extraction ofbeclomethasone dipropionate and triamcinolone acetonide. Deionized waterwas used for albuterol sulfate, cromolyn sodium, and DNase I. Foralbuterol MDIs, 0.5 ml of 1 N sodium hydroxide was added to the plateextract, which was used to convert the albuterol into the phenolateform.

Quantitation procedure. All drugs were quantitated by absorptionspectroscopy (Beckman DU640 spectrophotometer) relative to an externalstandard curve with the extraction solvent as the blank. Beclomethasonedipropionate and triamcinolone acetonide were quantitated by measuringthe absorption of the plate extracts at 238 nm Albuterol MDIs werequantified by measuring the absorption of the extracts at 243 mn, whilecromolyn sodium was quantitated using the absorption peak at 326 nm.DNase quantitation was done by a protein assay technique using Bio-Rad'smicrotiter plates (Bio-Rad Protein Assay Dye Reagent Concentrate)against a DNase calibration curve.

Calculation procedure. For each MDI, the mass of the drug in the stem(component −3), actuator (−2), induction port (−1) and plates (0-7) werequantified as described above. The Fine Particle Dose and Fine ParticleFraction was calculated according to the USP method referenced above.Throat deposition was defined as the mass of drug found in the inductionport and on plates 0 and 1. The mean mass aerodynamic diameters (MMAD)and geometric standard diameters (GSD) were evaluated by fitting theexperimental cumulative function with log-normal distribution by usingtwo-parameter fitting routine. The results of these experiments arepresented in subsequent examples.

XI Andersen Cascade Impactor Results for Albuterol MDI Formulations

The results of the cascade impactor test for two commercially availableformulations, Proventil HFA and Ventolin and an analogous spray driedhollow porous powder prepared according to Example III are tabulatedbelow. The Alliance formulation was prepared as described in Example IXabove. The actuator supplied with Proventil BFA (Key Pharmaceuticals)was used for assessing the performance of the hollow/porous particleMDIs. In all cases the actuator was washed and dried prior to eachAndersen Impactor test. The results are presented in Table IIimmediately below.

TABLE II Albuterol MDIs Throat Fine Fine Depo- particle Particle MMADsition, fraction, Dose, (GSD) μg % μg Proventil ®, HFA 2.6 ± 0.1 50.549.0 ± 0.7 48.5 ± 0.7 (3M Pharm.) (2.1 ± 0.3) 108 μg dose Ventolin ®,CFC 2.2 ± 0.2 58.9 43.5 ± 2.6 45.3 ± 3.3 (Glaxo Wellcome) (1.9 ± 0.1)108 μg dose Perforated 3.1 ± 0.2 14.9 79.3 ± 0.6 57.1 ± 5.7microstructures, HFA  (1.7 ± 0.01) (Alliance Pharm.) 60 μg dose

Proventil HFA and Ventolin were found to perform very similarly, with afine particle fraction of ˜45%, throat deposition of ˜55 μg, fineparticle dose of ˜47 μg, MMAD of ˜2.4 μm and GSD of ˜2.0. The MDIformulated with spray dried hollow porous particles had a substantiallyhigher fine particle fraction (˜80%), and significantly lower throatdeposition ( ˜15 g).

XII Andersen Cascade Impactor Results for Albuterol MDI Formulations:Effect of Suspension Concentration on Performance

Albuterol sulfate MDI dispersions prepared according to Examples III andIX were studied at different suspension concentrations to determine theeffect it may have upon fine particle fraction, MMAD, GSD, and fineparticle dose. MDIs containing 0.78% w/w., 0.46% w/w., 0.32% w/w., and0.25 % w/w spray dried hollow porous powders in HFA 134a were studied,and their results are tabulated and presented in Table III below.

TABLE III Spray-dried hollow porous albuterol sulfate Particles inHFA-134a MDI Fine particle Fine Particle wt % fraction, % Dose, μg MMADGSD 0.78 71 61.9 3.31 1.74 0.46 71 37.2 3.05 1.70 0.32 72 25.9 3.04 1.750.25 71 22.1 3.02 1.80

Similar performance was observed across the entire concentration rangefor the MDIs in terms of fine particle fraction, MMAD and GSD. A fineparticle dose ranging from 22.1 to nearly 62 μg was observed. Theseresults clearly demonstrate that a wide range of doses can be deliveredwithout any loss in fine particle fraction or any increase in throatdeposition. From a practical point of view this may be advantageous forboth low and high dose MDI applications.

XIII Andersen Cascade Impactor Results for Cromolyn Sodium MDIFormulations

The results of the cascade impactor tests for a commercially availableproduct (Intal, Rhone-Poulenc Rorer) and an analogous spray-dried hollowporous powder prepared according to Example IV and IX are shown below inTable IV.

TABLE IV Cromolyn Sodium MDIs Throat Fine Fine Depo- particle ParticleMMAD sition, fraction, Dose, (GSD) μg % μg Intal ®, CFC (n=4) 4.7 ± 0.5629 24.3 ± 2.1 202 ± 27 (Rhone Poulenc)  (1.9 ± 0.06) 800 μg dose Spraydried hollow 3.4 ± 0.2  97 67.3 ± 5.5 200 ± 11 porous powder, HFA (2.0 ±0.3) (Alliance) (n=3) 300 μg dose

The MDI formulated with perforated microstructures was found to havesuperior aerosol performance compared with Intal. At a comparable fineparticle dose, the spray dried cromolyn formulations possessed asubstantially higher fine particle fraction (˜67%), and significantlydecreased throat deposition (6-fold), and a smaller MMAD value. It isimportant to note that the effective delivery provided for by thepresent invention allowed for a fine particle dose that wasapproximately the same as the prior art comrnercial formulation eventhough the amount of perforated microstructures administered (300 μg)was roughly a third of the Intale® dose (800 μg) administered.

XIV Andersen Cascade Impactor Results for Beclomethasone DipropionateMDI Formulations

The results of cascade impactor tests for a commercially availableformulation (Vanceril, Schering Corp.) and a MDI formulation of ananalogous spray-dried hollow porous powder prepared according toExamples V and IX are listed below in Table V.

TABLE V Beclomethasone Dipropionate MDIs Throat Fine Fine Depo- particleParticle MMAD sition, fraction, Dose, (GSD) μg % μg Vanceril ®, CFC 3.4732 35 ± 2.1 17 ± 1.2 (n=4) (Schering) (2.29) 42 μg dose Perforated 3.7512 56.3 16 ± 0.7 microstructures, HFA (1.9)  (n=4) (Alliance) 28 μg dose

At an equivalent fine particle dose, the MDls formulated with spraydried hollowporous particles were found to have superior aerosolperformance compared with Vanceril. The spray dried beclomethasonedipropionate formulations possessed a substantially higher fine particlefraction (˜56% vs. 35%), and significantly lower throat deposition(˜3-fold) than Vanceril. The MMAD was found to be slightly higher forthe spray dried formulations.

XV Andersen Cascade Impactor Results for Triamcinolone Acetonide MDIFormulations

A comparison of a commercial formulation of triamcinolone acetonide(Azmacort, Rhone-Poulenc) and an MDI formulation of hollow porousparticles of TAA prepared according to Examples VI and IX are detailedbelow. Azinacort contains a built-in spacer device to limit steroiddeposition in the throat which causes local irritation and candidiasis.The results are shown in Table VI immediately below.

TABLE VI Triamcinolone Acetonide MDIs Throat Respir- Fine Dep- ableParticle MMAD Device sition fraction Dose μm μg μg % μg Azmacort ®, CFC6.0 133 42 11.5 23 (Rhone-Poulenc) 200 μg dose, (n=4) Perforated 3.4  1315 45.3 23 microstructures, HFA 50 μg dose, (Alliance) (n=4)

Roughly ⅔ of the initial dose of TAA in Azinacort was lost in the spacerdevice. Approximately ⅔ of the remaining dose was deposited in thethroat, with only 11.5% or 23 μg of the initial 200 μg available to thelung. In contrast, the perforated microstructures of the presentinvention administered without a spacer device deposited an equivalentdose with high efficiency, losing an order of magnitude less material inthe device and roughly three times less into the throat. Due to theincreased efficiency, four times less TAA is required to deliver therequired fine particle dose of 23 μg. These results show that thepresent formulations can eliminate the need for cumbersome spacerdevices in the delivery of steroids to the lung.

XVI Andersen Cascade Impactor Results for DNase I MDI Formulations

The inhalation properties of a MDI formulated as in Example IX withhollow porous particles of DNase I prepared according to Example VII wasassessed using an Andersen Cascade impactor. A fine particle fraction of76%, and MMAD of 3.31 μm were observed. The activity of the spray-driedDNase I powder was assessed for its ability to cleave DNA using gelelectrophoresis. No difference was observed between the neat andspray-dried DNase I particles.

XVII Effect of Powder Porosity on MDI Performance

In order to examine the effect powder porosity has upon the suspensionstability and aerodynamic diameter, MDIs were prepared with variouspreparations of perforated microstructures comprising gentamicinformulations as described in Example I. MDIs containing 0.48 wt % spraydried powders in HFA 134a were studied. As set forth in Example I, thespray dried powders exhibit varying porosity. The formulations werefilled in clear glass vials to allow for visual examination.

A strong dependence of the suspension stability and mean volume weightedaerodynamic diameter was observed as a function of PFC/PC ratio and/orporosity. The volume weighted mean aerodynamic diameter (VMAD) decreasedand suspension stability increased with increasing porosity. The powdersthat appeared solid and smooth by SEM and TEM techniques had the worstsuspension stability and largest mean aerodynamic diameter. MDIs whichwere formulated with highly porous and hollow perforated microstructureshad the greatest resistance to creaming and the smallest aerodynamicdiameters. The measured VMAD values for the dry powders produced inExample I are shown in Table VII immediately below.

TABLE VII PFC/PC Powder VMAD, μm 0 6.1 1.1 5.9 2.2 6.4 4.8 3.9 18.8 2.644.7 1.8

XVIII Comparison of Sedimentation Rates in Cromolyn Sodium Formulations

A comparison of the creaming rates of the commercial Intal formulation(Rhone-Poulenc Rorer) and spray-dried hollow porous particles formulatedin HFA-134a according to Examples IV and IX (i.e. see FIG. 2) is shownin FIGS. 3A to 3D. In each of the pictures, taken at 0 seconds, 30seconds, 60 seconds and two hours after shaking, the commercialformulation is on the left and the perforated microstructure dispersionformed accordance with the present invention is on the right. Whereasthe commercial Intal formulation shows sedimentation within 30 secondsof mixing, almost no sedimentation is noted in the spray-dried particlesafter 2 hours. Moreover, there was little sedimentation in perforatedmicrostructure formulation after 4 hours (not shown). This exampleclearly illustrates the balance in density which can be achieved whenthe hollow porous particles are filled with the suspension medium (i.e.in the formation of a homodispersion).

Those skilled in the art will further appreciate that, the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses only exemplaryembodiments thereof, it is to be understood that, other variations arecontemplated as being within the scope of the present invention.Accordingly, the present invention is not limited to the particularembodiments that have been described in detail herein. Rather, referenceshould be made to the appended claims as indicative of the scope andcontent of the invention.

What is claimed is:
 1. A stable respiratory dispersion for the pulmonarydelivery of one or more bioactive agents comprising a suspension mediumhaving dispersed therein a plurality of perforated microstructureshaving a mean aerodynamic diameter of less than 5 μm and comprising atleast one bioactive agent wherein said suspension medium comprises atleast one propellant and substanially permeates said perforatedmicrostructures wherein more than 30% of the average particle volume ofthe perforated microstructures is permeated by said suspension medium.2. The stable respiratory dispersion of claim 1, wherein said propellantcomprises a compound selected from the group consisting of1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane,perfluoroethane, monochlorodifluoromethane, 1,1-difluoroethane andcombinations thereof.
 3. The stable respiratory dispersion of claim 1wherein said propellant is a hydrofluoroalkane propellant.
 4. The stablerespiratory dispersion of claim 3 wherein said hydrofluoroalkanepropellant comprises 1,1,1,2-tetrrrluoroethane.
 5. The stablerespiratory dispersion of claim 3 wherein said hydrofluoroalkanepropellant comprises 1,1,1,2,3,3,3-heptafluoro-n-propane.
 6. The stablerespiratory dispersion of claim 1 wherein said perforatedmicrostructures comprise a surfactant.
 7. The stable respiratorydispersion of claim 6 wherein said surfactant is selected from the groupconsisting of phospholipids, nonionic detergents, nonionic blockcopolymers, ionic surfactants, biocompatible fluorinated surfactants andcombinations thereof.
 8. The stable dispersion of claim 6 wherein saidperforated microstructures comprise a poloxamer selected from the groupconsisting of poloxamer 188, poloxamer 407, and poloxamer
 338. 9. Thestable dispersion of claim 6 wherein said perforated microstructurescomprise oleic acid or its alkali salt.
 10. The stable respiratorydispersion of claim 6 wherein said surfactant comprises a lipid.
 11. Thestable respiratory dispersion of claim 10 wherein said lipid has a gelto liquid crystal phase transition greater than about 40° C.
 12. Thestable respiratory dispersion of claim 10 wherein said lipid is aphospholipid.
 13. The stable respiratory dispersion of claim 12 whereinsaid phospholipid is selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidyl-cholinebehenoylphosphatidylcholine, arachidoylphosphatidylcholine andcombinations thereof.
 14. The stable respiratory dispersion of claim 6wherein said perforated microstructures comprise greater than about 10%w/w surfactant.
 15. The stable respiratory dispersion of claim 14wherein said surfactant comprises a phospholipid.
 16. The stablerespiratory dispersion of claim 14 wherein said surfactant comprisesoleic acid or its alkali salt.
 17. The stable respiratory dispersion ofclaim 1 wherein said suspension medium and said perforatedmicrostructures have a refractive index differential of less than about0.4.
 18. The stable respiratory dispersion of claim 1 wherein saidsuspension medium and said perforated microstructures have a refractiveindex differential of less than about 0.3.
 19. The stable respiratorydispersion of claim 1 wherein said perforated microstructures comprisehollow porous microspheres.
 20. The stable respiratory dispersion ofclaim 19 wherein the microspheres comprise a surfactant.
 21. The stablerespiratory dispersion of claim 1 wherein the mean geometric diameter ofthe perforated microstructures is between 1 and 5 μm.
 22. The stablerespiratory dispersion of claim 1 wherein the mean geometric diameter ofthe perforated microstructures is between 0.5 and 5 μm.
 23. The stablerespiratory dispersion of claim 1 wherein the mean geometric diameter ofthe perforated microstructures is between 1 and 3 μm.
 24. The stablerespiratory dispersion of claim 1 wherein said bioactive agent has afine particle fraction following aerosolization of greater than 30% .25. The stable respiratory dispersion of claim 1 wherein said bioactiveagent has a fine particle fraction following aerosolization of greaterthan 50%.
 26. The stable respiratory dispersion of claim 1 wherein thedensity of the perforated microstructures permeated with the suspensionmedium substantially matches that of the suspension medium.
 27. Thestable respiratory dispersion of claim 1 wherein said bioactive agent isselected from the group consisting of antiallergics, bronchodilators,pulmonary lung surfactants, analgesics, antibiotics, antiinfectives,leukotriene inhibitors or antagonists, antihistamine,antiinflammatories, antineoplastics, antocholinergics, anesthetics,anti-tuberculars, imaging agents, cardiovascular agents, enzymes,steroids, genetic material, viral vectors, antisense agents, proteins,peptides, and combinations thereof.
 28. The stable respiratorydispersion of claim 1 wherein said bioactive agents are selected fromthe group consisting of steroids, bronchodilators and peptides.
 29. Thestable respiratory dispersion of claim 1 wherein said bioactive agentsare selected from the group consisting of budesonide, fluticasonepropionate, salrieterol, formoterol, gentamicin, LHRH, and DNase.
 30. Amethod for forming a stabilized respiratory dispersion comprising thesteps of: combining a plurality of perforated microstructures having amean aerodynamic diameter of less than 5 μm and comprising at least onebioactive agent with a predetermined volume of suspension mediumcomprising at least one propellant to provide a respiratory blendwherein said suspension medium pernates said microstructures whereinmore than 30% of the average particle volume of the perforatedmicrostructures is permeated by said suspension medium; and mixing saidrespiratory blend to provide a substantidly homogeneous respiratorydispersion.
 31. A respiratory dispersion formed according to the methodof claim
 30. 32. The method of claim 30 further comprising the step ofspray drying an oil-in-water emulsion to provide said perforatedmicrostructures wherein the disperse phase of said emulsion comprises afluorochemical.
 33. A respiratory dispersion formed according to themethod of claim
 32. 34. The method of claim 32 wherein saidfluorochemical has a boiling point of greater than 60° C.
 35. The methodof claim 30, wherein said propellant comprises a compound selected fromthe group consisting of 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane, perfluoroethane,monochlorodifluoromethane, 1,1-difluoroethane and combinations thereof.36. The method of claim wherein said propellant comprises ahydrofluoroalkane propellant.
 37. The method of claim 36 wherein saidhydrofluoroalkane propellant comprises 1,1,1,2-tetrafluorethane.
 38. Themethod of claim 36 wherein said hydrofluoroalkane propellant comprises1,1,1,2,3,3,3-heptafluoro-n-propane.
 39. The method of claim 30 whereinsaid perforated microstructures comprise a surfactant.
 40. The method ofclaim 39 wherein said surfactant is selected from the group consistingof phospholipids, nonionic detergents, nonionic block copolymers, ionicsurfactants, biocompatible fluorinated surfactants and combinationsthereof.
 41. The method of claim 39 wherein said surfactant comprises alipid.
 42. The method of claim 41 wherein said lipid has a gel to liquidcrystal phase transition greater than 40° C.
 43. The method of claim 41wherein said lipid is a phospholipid.
 44. The method of claim 43 whereinsaid phospholipid is selected from the group consisting ofdilauroylphosphatidylcholine, dioleyphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine andcombinations thereof.
 45. The method of claim 39 wherein said perforatedmicrostructures comprise greater than about 10% w/w surfactal.
 46. Themethod of claim 36 wherein said suspension medium and said perforatedmicrostrures have a refractive index differential of less than about0.4.
 47. The method of claim 30 wherein said perforated microstructurescomprise hollow microspheres.
 48. The method of claim 30 wherein themean geometric diameter of the perforated microstructures is between 1and 5 μm.
 49. The method of claim 30 wherein the mean aerodynamicdiameter of the perforated microstructures is between 0.5 and 5 μm. 50.The method of claim 30 wherein said bioactive agent has a fine particlefraction following aerosolization of greater than 30%.
 51. The method ofclaim 30 wherein the density of the suspended microstructures permeatedwith the suspension medium substantially matches that of the suspensionmedium.
 52. The method of claim 30 wherein said bioactive agent isselected from the group consisting of antiallergics, bronchodilators,pulmonary lung surfactants, analgesics, antibiotics, leukotrieneinhibitors or antagonists, antihistamines, antiinflammatories,antineoplastics, antcholinergics, anesthetics, anti-tuberculars, imagingagents, cardiovascular agents, enzymes, steroids, genetic material,viral vectors, antisense agents, proteins, peptides and combinationsthereof.
 53. A method for stabilizing a dispersion by reducingattractive van der Waals forces comprising providing a plurality ofperforated microstructures having a mean aerodynamic diameter of lessthan 5 μm and; combining the microstructures with a suspension mediumcomprising at least one propellant wherein the wherein more than 30% ofthe average particle volume of the perforated microstrures is permeatedby said suspension medium.
 54. A dispersion formed according to themethod of claim
 53. 55. The method of claim 53 wherein said refractiveindex differential value is less than 0.3.
 56. The method of claim 53wherein said propellant comprises a compound selected from the groupconsisting of 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane, perfluoroethane,monochlorodifluoromethane, 1,1-difluoroethane and combinations thereof.57. The method of claim 53 wherein said propellant comprises ahydrofluoroakane propellant.
 58. The method of claim 57 wherein saidhydrofluoroalkane propellant comprises 1,1,1,2-tetafluoroethane.
 59. Themethod of claim 57 wherein said hydrofluoroalkane propellant comprises1,1,1,2,3,3,3-heptafluoro-n-propane.
 60. The method of claim 53 whereinsaid perforated microstructures comprise a surfant.
 61. The method ofclaim 60 wherein said surfactant is selected from the group consistingof phospholipids, nonionic detergents, nonionic block copolymers, ionicsurfactants, biocompatible fluorinated surfactants and combinationsthereof.
 62. The method of claim 60 wherein said surfactant comprises alipid.
 63. The method of claim 62 wherein said lipid has a gel to liquidcrystal phase transition greater than 40° C.
 64. The method of claim 62wherein said lipid is a phospholipid.
 65. The method of claim 64 whereinsaid phospholipid is selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine andcombinations thereof.
 66. The method of claim 53 wherein said perforatedmicrostructures comprise greater than 10% w/w surfactant.
 67. The methodof claim 53 wherein said perforated microstructures comprise hollowporous microspheres.
 68. The method of claim 53 wherein the meangeometric diameter of the perforated microstructures is between 0.5 and5 μm.
 69. The method of claim 53 wherein the mean aerodynamic diameterof the perforated microstructures is between 0.5 and 5 μm.
 70. Themethod of claim 53 the density of the suspended microstructes permeatedwith the suspension medium substantially matches that of the suspensionmedium.
 71. The method of claim 53 wherein said perforatedmicrostructures comprise a bioactive agent selected from the groupconsisting of antiallergics, bronchodilators, pulmonary lungsurfactants, analgesics, antibiotics, leukotriene inhibitors orantagonists, antihistamines, antiinflammatories, antineoplastics,antcholinergics, anesthetics, anti-tuberculars, imaging agents,cardiovascular agents, enzymes, steroids, genetic material, viralvectors, antisense agents, proteins, peptides and combinations thereof.72. A respiratory dispersion for the pulmonary delivery of one or morebioactive agents comprising a suspension medium having dispersed thereina plurality of microparticles having a mean aerodynamic diameter of lessthan 5 μm and comprising greater than about 20% w/w surfactant and atleast one bioactive agent wherein said suspension medium comprises atleast one propellant.
 73. The respiratory dispersion of claim 72 whereinsaid dispersed microparticles comprise greater than about 30% w/wsurfactant.
 74. The respiratory dispersion of claim 72 wherein saidpropellant comprises a compound selected from the group consisting of1,1,1,2-etrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane,perfluoroethane, monochlorodifluoromethane, 1,1-difluoroethane andcombinations thereof.
 75. The respiratory dispersion of claim 72 whereinsaid propellant is a hydrofluoroalkane propellant.
 76. The respiratorydispersion of claim 75 wherein said hydrofluoroalkane propellantcomprises 1,1,1,2-tetrafluoroethane.
 77. The respiratory dispersion ofclaim 72 wherein said surfactant is selected from the group consistingof phospholipids, nonionic detergents, nonionic block copolymers, ionicsurfactants, biocompatible fluorinated surfactants and combinationsthereof.
 78. The respiratory dispersion of claim 72 wherein saidsurfactant comprises a lipid.
 79. The respiratory dispersion of claim 78wherein said lipid has a gel to liquid crystal phase transition greaterthan about 40° C.
 80. The respiratory dispersion of claim 78 whereinsaid lipid is a phospholipid.
 81. The respiratory dispersion of claim 80wherein said phospholipid is selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine andcombinations thereof.
 82. The respiratory dispersion of claim 72 whereinsaid microparticles comprise perforated microstructures.
 83. Therespiratory dispersion of claim 82 wherein said perforatedmicrostructures comprise hollow porous microspheres.
 84. The respiratorydispersion of claim 83 wherein said hollow porous microspheres have amean aerodynamic diameter between 0.5 to 5 μm.
 85. The respiratorydispersion of claim 72 wherein the mean geometric diameter of themicroparticles is between 1 and 5 μm.
 86. The respiratory dispersion ofclaim 72 wherein said bioactive agent is selected from the groupconsisting of antiallergics, bronchodilators, pulmonary lungsurfactants, analgesics, antibiotics, leukotriene inhibitors orantagonists, antihistamines, antiinflammatories, antineoplastics,antcholinergics, anesthetics, anti-tuberculars, imaging agents,cardiovascular agents, enzymes, steroids, genetic material, viralvectors, antisense agents, proteins, peptides and combinations thereof.87. The stable respiratory dispersion of claim 72 wherein said bioactiveagents are selected from the group consisting of budesonide, fluticasonepropionate, salmeterol, formoterol and DNase.
 88. The stable respiratorydispersion of claim 1 further comprising a creaming or sedimentationtime greater than 1 minute.
 89. The stable respiratory dispersion ofclaim 80 wherein the creaming or sedimentation time is greater than 30minutes.
 90. The stable respiratory dispersion of claim 1 wherein thegeometric mean diameter of the microstructures is between 1-30 μm. 91.The stable respiratory dispersion of claim 27 wherein the bioactiveagent is an antiinfective selected from the group consisting ofcephalosporins, macrolides, quinolines, penicillins, streptomycin,sulphonamides, tetracyclines, and pentamidine.
 92. The respiratorydispersion of claim 82 wherein said perforated microstructures comprisea poloxamer selected from the group consisting of poloxamer 188,poloxamine 407 and poloxamer
 338. 93. The respiratory dispension ofclaim 82 wherein said perforate microstructures comprise oleic acid orits alkali salt.